Quantum Chemical and Biological Insights into Redox Activity of Metallacarborane Complexes in Cancer Cells.

A relationship between the electronic properties of metal ions in metallacarboranes and their ability to modulate mitochondrial oxidase activity and membrane hyperpolarization in cancer cells was demonstrated. Quantum chemistry methods, including DFT and molecular dynamics simulations, were used to understand the oxidized and reduced forms of metallacarboranes and their intramolecular rotatory behavior. According to the low-spin assumption for metal ions, the intramolecular oscillations of cluster ligands in metallacarboranes are significantly influenced by the type of metal and correspond to the cellular uptake of these complexes in vitro. In particular, the low-spin iron compound may be a new xenogeneic booster of redox homeostasis in cancer cells resistant to cisplatin, which induces metabolic 'exhaustion' of cancer cells and their death.

Early Stage In Vitro Bioprofiling of Potential Low-Molecular-Weight Organoboron Compounds for Boron Neutron Capture Therapy (BNCT)-Proposal for a Guide.

Given the renewed interest in boron neutron capture therapy (BNCT) and the intensified search for improved boron carriers, as well as the difficulties of coherently comparing the carriers described so far, it seems necessary to define a basic set of assays and standardized methods to be used in the early stages of boron carrier development in vitro. The selection of assays and corresponding methods is based on the practical experience of the authors and is certainly not exhaustive, but open to discussion. The proposed tests/characteristics: Solubility, lipophilicity, stability, cytotoxicity, and cellular uptake apply to both low molecular weight (up to 500 Da) and high molecular weight (5000 Da and more) boron carriers. However, the specific methods have been selected primarily for low molecular weight boron carriers; in the case of high molecular weight compounds, some of the methods may need to be adapted.

Normalized Multipotential Redox Coding of DNA Bases for Determination of Total Nucleotide Composition.

The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

Metallacarborane Synthons for Molecular Construction-Oligofunctionalization of Cobalt Bis(1,2-dicarbollide) on Boron and Carbon Atoms with Extendable Ligands.

The exploitation of metallacarboranes' potential in various fields of research and practical applications requires the availability of convenient and versatile methods for their functionalization with various functional moieties and/or linkers of different types and lengths. Herein, we report a study on cobalt bis(1,2-dicarbollide) functionalization at 8,8'-boron atoms with different hetero-bifunctional moieties possessing a protected hydroxyl function allowing further modification after deprotection. Moreover, an approach to the synthesis of three and four functionalized metallacarboranes, at boron and carbon atoms simultaneously via additional functionalization at carbon to obtain derivatives carrying three or four rationally oriented and distinct reactive surfaces, is described.

Intramolecular rotations and electronic states of iron in the iron bis(dicarbollide) complex Fe[(C2B9H11)2] studied by a 57Fe nuclear probe and computational methods.

Mössbauer spectroscopy of iron(III) bis(dicarbollide) (1) and its adduct (2) revealed low spin FeIII in 1 and surprisingly FeII in 2. In 1, the (C2B9H11) groups rotate at room temperature with a frequency of 107 Hz, getting across the energy barrier of 24 meV. Numerical simulations showed a gradient of electric charge in 2, which may explain the FeII-like character in 2.

Antiviral Effect of Nonfunctionalized Gold Nanoparticles against Herpes Simplex Virus Type-1 (HSV-1) and Possible Contribution of Near-Field Interaction Mechanism.

The antiviral activity of nonfunctionalized gold nanoparticles (AuNPs) against herpes simplex virus type-1 (HSV-1) in vitro was revealed in this study. We found that AuNPs are capable of reducing the cytopathic effect (CPE) of HSV-1 in Vero cells in a dose- and time-dependent manner when used in pretreatment mode. The demonstrated antiviral activity was within the nontoxic concentration range of AuNPs. Interestingly, we noted that nanoparticles with smaller sizes reduced the CPE of HSV-1 more effectively than larger ones. The observed phenomenon can be tentatively explained by the near-field action of nanoparticles at the virus envelope. These results show that AuNPs can be considered as potential candidates for the treatment of HSV-1 infections.

Interaction of Adenosine, Modified Using Carborane Clusters, with Ovarian Cancer Cells: A New Anticancer Approach against Chemoresistance.

Platinum compounds remain the first-line drugs for the treatment of most lethal gynecological malignancies and ovarian cancers. Acquired platinum resistance remains a major challenge in gynecological oncology. Considering the unique physicochemical properties of the metallacarboranes modifier and the significant role of nucleoside derivatives as anticancer antimetabolites, we designed and synthesized a set of adenosine conjugates with metallacarboranes containing iron, cobalt, or chromium as semi-abiotic compounds that influence the cisplatin sensitivity of ovarian cancer cells. Adherent cultures of ovarian carcinoma cell lines and multicellular spheroids, ranging from sensitive to highly resistant including experimental cell lines "not responding" to platinum drugs were used. Iron-containing metallacarborane conjugates showed the best anticancer activity, especially against resistant cells. Compound modified at the C2' nucleoside position showed the best activity in resistant cancer cells and highly resistant cancer spheroids exposed to cisplatin, increasing cell cycle arrest, apoptosis or necrosis, and reactive oxygen species production. Moreover, it showed high cellular accumulation and did not induce cross-resistance to cisplatin, carboplatin, doxorubicin, paclitaxel, or gemcitabine in long-term cultures. The reference nido-carborane derivative (no metal ions) and unmodified nucleosides were not as effective. These findings indicate that metallacarborane modification of adenosine may sensitize ovarian cancer cells to cisplatin in combination treatment.

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists.

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A1, A2A, A2B and A3 adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A3 AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N6-(3-phenylpropyl)adenosine (18), N6-(3-phenylpropyl)-2-chloroadenosine (24) and N6-(3-phenylpropyl)adenosine (40) showed nanomolar A3 affinity (Ki 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Insight into the expression of RIG-I-like receptors in human third trimester placentas following ex vivo cytomegalovirus or vesicular stomatitis virus infection.

A viral infection is detected through germline-encoded pattern-recognition receptors (PRRs) leading to the production of interferons (IFNs) and proinflammatory cytokines. The objective of this study was to investigate the expression of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) in response to viral infection and the selected cytokine responses in the human term placenta. Placental villi and decidual explants were infected with human cytomegalovirus (CMV) or vesicular stomatitis virus (VSV) and cultured ex vivo to study viral infection. To evaluate DDX58 (RIG-I), IFIH1 (MDA5), and DHX58 (LGP2) expression, quantitative real-time PCR (qRT-PCR) was used. The expression of RLRs was detected by Western blotting. Cytokine and chemokine production, as well as RLR protein levels, were quantified using ELISA. The increased expression of both RIG-I and MDA5 and the enhanced secretion of IFN-ß were observed in response to VSV infection compared to mock-infected tissues. CMV infection resulted in higher transcript levels of DDX58 and IFIH1, while no changes in the cytokine production were observed. Our results indicate that RIG-I and MDA5 are specifically expressed in chorionic villi and deciduae in response to VSV infection. These findings suggest that RLRs may play a key role in pathogen recognition and the immune response against intrauterine viral transmission.

Comparative study of the effects of ortho-, meta- and para-carboranes (C2B10H12) on the physicochemical properties, cytotoxicity and antiviral activity of uridine and 2'-deoxyuridine boron cluster conjugates.

In this study, a series of uridine (U) and 2'-deoxyuridine (dU) conjugates containing an isomeric ortho-, meta- or para-carborane cluster (C2B10H12) attached at C-5 through an ethynyl linker were synthesized. The effect of carborane cluster isomerism on the conjugate syn/anti conformation, molar extinction coefficient, lipophilicity, susceptibility to phosphorylation (by TK1, TK2 and dCK), cytotoxicity and antiviral activity was evaluated. A strong effect of the boron cluster modification on the syn/anti equilibrium of the modified nucleosides was observed. An increase in lipophilicity compared with unmodified U and dU, especially for conjugates bearing a para-carborane cluster, was detected. Furthermore a pronounced and differential influence of the boron cluster modification on the electronic properties of the nucleobase chromophore was observed. The obtained conjugates have low or medium toxicity toward several cell lines, are phosphorylated fairly well by TK1 and are poor or not substrates for dCK. Furthermore, the conjugates preferentially inhibit HCMV replication with an SI index as high as 22 for the ortho-carborane derivative of U and more than 180 for the para-carborane derivative of dU.

Comparative study of inorganic, boron-rich cluster and organic, phenyl adenosine modifications: synthesis and properties.

Background: Nucleoside analogs are important class of chemotherapeutics. One of the original openings in the nucleoside medicinal chemistry was derivatives comprising a boron cluster component. Results: A series of adenosine derivative pairs containing inorganic boron cluster or alternatively its mimic, organic phenyl modification were synthesized and their physicochemical and biological properties compared. Marked effects of boron clusters, which are qualitatively and quantitatively different from the phenyl group effects, were detected. The studied characteristics include syn/anti conformation, lipophilicity, cytotoxicity and antiviral activity, as well as phosphorylation by adenosine kinase. Conclusion: The obtained results demonstrate usefulness of the boron clusters for tuning properties of biomolecules and prove their potential as modifying units in design of future therapeutics based on nucleoside structures.

Raman cell imaging with boron cluster molecules conjugated with biomolecules.

Raman spectroscopic measurements and theoretical calculation revealed that the Raman bands corresponding to the B-H stretching vibrations of two types of simple icosahedral boron clusters, ortho-carborane 3 and closo-dodecaborate 4 appeared at approximately 2450-2700 cm-1, and did not overlap with those of cellular components. Although ortho-carborane 3 possesses a possible property as a Raman probe, it was difficult to measure Raman imaging in the cell due to its poor water solubility. In fact, ortho-carborane derivative 6, which internally has an alkyne moiety, exhibited very weak Raman signals of the C[triple bond, length as m-dash]C stretching and the B-H stretching vibrations were barely detected at a 400 ppm boron concentration in HeLa cells. In contrast, closo-dodecaborate derivatives such as BSH (5) were found to be a potential Raman imaging probe cluster for target molecules in the cell. BSH-conjugated cholesterol 7 (BSH-Chol) was synthesized and used in Raman imaging in cells. Raman imaging and spectral analysis revealed that BSH-based Raman tags provide a versatile platform for quantitative Raman imaging.

Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives.

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10-12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10-12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.

DNA Modified with Boron⁻Metal Cluster Complexes [M(C2B9H11)2]-Synthesis, Properties, and Applications.

Together with tremendous progress in biotechnology, nucleic acids, while retaining their status as "molecules of life", are becoming "molecular wires", materials for the construction of molecular structures at the junction between the biological and abiotic worlds. Herein, we present an overview of the approaches for incorporating metal centers into nucleic acids based on metal⁻boron cluster complexes (metallacarboranes) as the metal carriers. The methods are modular and versatile, allowing practical access to innovative metal-containing DNA for various applications, such as nucleic acid therapeutics, electrochemical biosensors, infrared-sensitive probes, and building blocks for nanoconstruction.

Comparative Study of Carborane- and Phenyl-Modified Adenosine Derivatives as Ligands for the A2A and A3 Adenosine Receptors Based on a Rigid in Silico Docking and Radioligand Replacement Assay.

Adenosine receptors are involved in many physiological processes and pathological conditions and are therefore attractive therapeutic targets. To identify new types of effective ligands for these receptors, a library of adenosine derivatives bearing a boron cluster or phenyl group in the same position was designed. The ligands were screened in silico to determine their calculated affinities for the A2A and A3 adenosine receptors. An virtual screening protocol based on the PatchDock web server was developed. In the first screening phase, the effects of the functional group (organic or inorganic modulator) on the adenosine ligand affinity for the receptors were determined. Then, the lead compounds were identified for each receptor in the second virtual screening phase. Two pairs of the most promising ligands, compounds 3 and 4, and two ligands with lower affinity scores (compounds 11 and 12, one with a boron cluster and one with a phenyl group) were synthesized and tested in a radioligand replacement assay for affinity to the A2A and A3 receptors. A reasonable correlation of in silico and biological assay results was observed. In addition, the effects of a phenyl group and boron cluster, which is new adenosine modifiers, on the adenosine ligand binding were compared.

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.

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.