Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed

Docking of indolo- and pyrrolo-pyrimidines to DNA. New DNA-interactive polycycles from amino-indoles/pyrroles and BMMA.

New indolo- and pyrrolo-pyrimidines of type 1-4 were studied for their ability to form stable complexes with DNA fragments. The calculated free energies of binding were found in the range -8.39 ÷ -16.72 Kcal/mol. The docking studies revealed a common binding mode with the chromophore intercalated between GC base pairs whereas the side chain lies along the minor groove

Enhancing the cellular uptake of Py–Im polyamides through next-generation aryl turns

Pyrrole–imidazole (Py–Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein–DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5′-WGWWCW-3′ (W = A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py–Im polyamides that will prove valuable for future applications in which biological potency is essential

Programing strand displacement reaction pathways using small molecular DNA binders

DNA has been used in nature as carriers of heredity information for billions of years. The last four decades have witnessed the success of DNA nanotechnology, an interdisciplinary research area in which DNA is used as a synthetic engineering tool rather than a carrier of genetic information. The growth of DNA nanotechnology crosses the boundaries between physics, chemistry, biology and computer science and enables DNA to function as an electronic component, substrate, drug delivery vector and data storage unit. The hybridization of DNA strictly follows the by Watson-Crick rule; thus, DNA base pairs are the most reliable and predictable building block in the true nanometer range. New methods and designs for controlling DNA hybridization have always provided the most essential momentum for the development of DNA nanotechnology. When small molecules bind to the double helical structure of DNA, either through intercalation or minor groove binding, the stability and functionality of DNA may be significantly altered, which is a fundamental basis for many therapeutic and sensing applications. Herein, we reveal, for the first time, that small molecular DNA binders may also be used to program the reaction pathways of toehold-mediated DNA strand displacement, an elementary building block in DNA nanotechnology

The Stargazin-Related Protein {gamma}7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including CaV2.2

The role(s) of the novel stargazin-like {gamma}-subunit proteins remain controversial. We have shown previously that the neuron-specific {gamma}7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of {gamma}7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of {gamma}7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous {gamma}7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed {gamma}7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of {gamma}7 is essential for all its effects, and we show that {gamma}7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa, and this enhancement is prevented by a concentration of {gamma}7 that alone has no effect on IBa. The effect of {gamma}7 is selective for certain mRNAs because it had no effect on {alpha}2{delta}-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that {gamma}7 plays a role in stabilizing CaV2.2 mRNA

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition

Biochemical Effects of Irradiation on Larvae of Schistosoma mansoni, Volume II

Abstract Not Provided

Diastereomeric bactericidal effect of Ru(phenanthroline)(2)dipyridophenazine

Metal susceptibility assays and spot plating were used to investigate the antimicrobial activity of enantiopure [Ru(phen)(2)dppz](2+) (phen =1,10-phenanthroline and dppz = dipyrido[3,2-a:2 ',3 '-c]phenazine) and [-bidppz(phen)(4)Ru-2](4+) (bidppz =11,11 '-bis(dipyrido[3,2-a:2 ',3 '-c]phenazinyl)), on Gram-negative Escherichia coli and Gram-positive Bacillus subtilis as bacterial models. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined for both complexes: while [-bidppz(phen)(4)Ru-2](4+) only showed a bactericidal effect at the highest concentrations tested, the antimicrobial activity of [Ru(phen)(2)dppz](2+) against B. subtilis was comparable to that of tetracyline. In addition, the -enantiomer of [Ru(phen)(2)dppz](2+) showed a 2-fold higher bacteriostatic and bactericidal effect compared to the -enantiomer. This was in accordance with the enantiomers relative binding affinity for DNA, thus strongly indicating DNA binding as the mode of action

Coupling Tumor Necrosis Factor-α with αV Integrin Ligands Improves Its Antineoplastic Activity

Despite the impressive results obtained in animal models, the clinical use of tumor necrosis factor-α (TNF) as an anticancer drug is limited by severe toxicity. We have shown previously that targeted delivery of TNF to aminopeptidase N (CD13), a marker of angiogenic vessels, improved the therapeutic index of this cytokine in tumor-bearing mice. To assess whether the vascular-targeting approach could be extended to other markers of tumor blood vessels, in this work, we have fused TNF with the ACDCRGDCFCG peptide, a ligand of αV integrins by recombinant DNA technology. We have found that subnanogram doses of this conjugate are sufficient to induce antitumor effects in tumor-bearing mice when combined with melphalan, a chemotherapeutic drug. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the Arg-Gly-Asp moiety interacts with cell adhesion receptors, including αVβ3 integrin, as originally postulated. In addition, ACGDRGDCFCG-mouse TNF conjugate induced cytotoxic effects in standard cytolytic assays, implying that ACGDRGDCFCG-mouse TNF conjugate can also bind TNF receptors and trigger death signals. These results indicate that coupling TNF with αV integrin ligands improves its antineoplastic activity and supports the concept that vascular targeting is a strategy potentially applicable to different endothelial markers, not limited to CD13