A Convenient Oligonucleotide Conjugation via Tandem Staudinger Reaction and Amide Bond Formation at the Internucleotidic Phosphate Position.

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5'-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide.

Antitumor efficacy of multi-target in situ vaccinations with CpG oligodeoxynucleotides, anti-OX40, anti-PD1 antibodies, and aptamers.

To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.

New Hybrid Compounds Combining Fragments of Usnic Acid and Thioether Are Inhibitors of Human Enzymes TDP1, TDP2 and PARP1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3' phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA-TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4-25.2 µM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.

Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

A New Antisense Phosphoryl Guanidine Oligo-2'-O-Methylribonucleotide Penetrates Into Intracellular Mycobacteria and Suppresses Target Gene Expression.

The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2'-O-methylribonucleotides (2'-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2'-OMe PGO in mycobacteria. The results indicated that 2'-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2'-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2'-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2'-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2'-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.

Neutral and Negatively Charged Phosphate Modifications Altering Thermal Stability, Kinetics of Formation and Monovalent Ion Dependence of DNA G-Quadruplexes.

The effect of phosphate group modifications on formation and properties of G-quadruplexes (G4s) has not been investigated in detail. Here, we evaluated the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes formed by oligodeoxynucleotides d(G4 T), d(TG4 T) and d(TG5 T), in which all phosphates were replaced with N-methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups resulting in either negatively charged or neutral DNA sequences, respectively. We established that all modified sequences were able to form G-quadruplexes of parallel topology; however, the presence of modifications led to a decrease in thermal stability relative to unmodified G4s. In contrast to negatively charged G4s, assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions, and was independent of the salt concentration used. Formation of mixed G4s composed of both native and neutral G-rich strands has been detected using native gel electrophoresis, size-exclusion chromatography and ESI-MS. In summary, our results indicate that the phosphate modifications studied are compatible with G-quadruplex formation, which could be used for the design of biologically active compounds.

Biological evaluation of tetracationic compounds based on two 1,4-diazabicyclo[2.2.2]octane moieties connected by different linkers.

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2h. Furthermore, compounds 1a-c with aromatic linkers and compound 1e showed the highest antiviral activity.

Synthesis and structure-activity relationship of novel 1,4-diazabicyclo[2.2.2]octane derivatives as potent antimicrobial agents.

A series of new quaternary 1,4-diazabicyclo[2.2.2]octane derivatives was synthesized and evaluated for activity against several strains of both Gram positive and Gram negative bacteria and one strain of fungus under different inoculum size. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six species of microorganisms were tested. Results show a clear structure-activity relationship between alkyl chain length of substitutions of 1,4-diazabicyclo[2.2.2]octane tertiary amine sites and antimicrobial activity. In the case of compounds 4a-4k, MIC was found to decrease with the increase of the alkyl chain length from ethyl to dodecyl and then to increase at higher chain length (n > 14). The MIC values were found to be low for the compounds 4f and 4g with alkyl chains ranging from 10 to 12 carbons in length (1.6 µg/ml) and were comparable to the reference drug Ciprofloxacin. Also, time-kill assay was performed to examine the bactericidal kinetics. Results indicated that 4f and 4g had rapid killing effects against Staphylococcus aureus, and eliminated 100% of the initial inoculum of bacteria in 2.5 h at the concentration of 10 µg/ml. In addition, compound 4g eliminate more than 99.9% of the initial inoculum of Ps. aeruginosa after 2.5 h of interaction but the activity of compound 4f against this species seems to be weak. Thus, 4g had strong bactericidal activity and could rapidly kill Gram positive S. aureus, as well as Gram negative Ps. aeruginosa at low and high inoculum size.

Molecular design of artificial ribonucleases using electrostatic interaction.

A number of small organic ribonucleases have been synthesized with rigid polycationic structures containing an aromatic framework with two residues of bis-quaternary salts of 1,4-diazabicyclo[2.2.2]octane (DABCO) bearing various substituents. The compounds carrying positively charged groups connected via rigid linker are expected to bend the sugar-phosphate backbone and can stimulate the intramolecular phosphoester transfer reaction.