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Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

The aim of this research is to obtain new data about the complexation between β-cyclodextrin (β-CD) and benzoic acid (BA) as a model reaction of the complex formation of hydrophobic molecules with cyclodextrins (CDs) in various media. This research may help developing cyclodextrin-based pharmaceutical formulations through the choice of the appropriate solvent mixture that may be employed in the industrial application aiming to control the reactions/processes in liquid phase. In this paper, NMR results for the molecular complex formation between BA and β-CD ([BA⊂β-CD]) in D 2 O-DMSO- d 6 and in D 2 O-EtOH have shown that the stability of the complex in the H 2 O-DMSO- d 6 varies within the experimental error, while decreases in H 2 O-EtOH. Changes in the Gibbs energy of BA resolvation in water and water-dimethylsulfoxide mixtures have been obtained and have been used in the analysis of the reagent solvation contributions into the Gibbs energy changes of the [BA⊂β-CD] molecular complex formation. Quantum chemical calculations of the interaction energy between β-CD and BA as well as the structure of the [BA⊂β-CD] complex and the energy of β-CD and BA interaction in vacuum and in the medium of water, methanol and dimethylsulfoxide solvents are carried out. The stability of [BA⊂β-CD] complex in H 2 O-EtOH and H 2 O-DMSO solvents, obtained by different methods, are compared. The thermodynamic parameters of the [BA⊂β-CD] molecular complexation as well as the reagent solvation contributions in H 2 O-EtOH and H 2 O-DMSO mixtures were analyzed by the solvation-thermodynamic approach.

Chemotherapy represents the most applied approach to cancer treatment. Owing to the frequent onset of chemoresistance and tumor relapses, there is an urgent need to discover novel and more effective anticancer drugs. In the search for therapeutic alternatives to treat the cancer disease, a series of hybrid pyrazolo[3,4- d ]pyrimidin-4( 5H )-ones tethered with hydrazide-hydrazones, 5a - h , was synthesized from condensation reaction of pyrazolopyrimidinone-hydrazide 4 with a series of arylaldehydes in ethanol, in acid catalysis. In vitro assessment of antiproliferative effects against MCF-7 breast cancer cells, unveiled that 5a , 5e , 5g , and 5h were the most effective compounds of the series and exerted their cytotoxic activity through apoptosis induction and G0/G1 phase cell-cycle arrest. To explore their mechanism at a molecular level, 5a , 5e , 5g , and 5h were evaluated for their binding interactions with two well-known anticancer targets, namely the epidermal growth factor receptor (EGFR) and the G-quadruplex DNA structures. Molecular docking simulations highlighted high binding affinity of 5a , 5e , 5g , and 5h towards EGFR. Circular dichroism (CD) experiments suggested 5a as a stabilizer agent of the G-quadruplex from the Kirsten ras (KRAS) oncogene promoter. In the light of these findings, we propose the pyrazolo-pyrimidinone scaffold bearing a hydrazide-hydrazone moiety as a lead skeleton for designing novel anticancer compounds.

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5'-AGGGCGGTGTGGGAATAGGGAA-3' ( KRAS 22RT) and 5'-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3' ( KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.

Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC)·poly(dG-dC) and the poly(dA-dT)·poly(dA-dT), by means of UV-Vis and circular dichroism (CD) spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues., Competing Interests: The authors declare no conflict of interest.

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.