G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4’s including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.

Resveratrol is a naturally occurring stilbene which has shown promising results as treatment for several neurodegenerative diseases. However, its application is limited due to its low efficacy and bioavailability. Here, we have designed and synthesized alkylated resveratrol prodrugs combining structural modification to improve antioxidant and anti-inflammatory properties and the preparation of prodrugs to extend drug bioavailability. For comparison we also studied resveratrol prodrugs and alkylated resveratrol derivatives. Methylated and butylated resveratrol derivatives showed the best in vitro neuroprotective and anti-inflammatory activity. The glucosyl- and glucosyl-acyl- prodrugs of these derivatives showed lower toxicity on zebra fish embryo. When neuroprotection was examined on pentylenetetrazole challenged zebra fish, they were capable of reverting neuronal damage but to a lower extent than resveratrol. Nevertheless, 3-O-(6'-O-octanoyl)-β-d-glucopyranoside resveratrol (compound 8) recovered AChE activity over 100% whereas resveratrol only up to 92%. In a 3-nitropropionic acid mice model of Huntington's disease, resveratrol derivative 8 delayed the onset and reduced the severity of HD-like symptoms, by improving locomotor activity and protecting against weight loss. Its effects involved an equal antioxidant but better anti-inflammatory profile than resveratrol as shown by SOD2 expression in brain tissue and circulating levels of IL-6 (11 vs 18 pg/mL), respectively. Finally, the octanoyl chain in compound 8 could be playing a role in inflammation and neuronal development indicating it could be acting as a double-drug, instead of as a prodrug.

Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. We found out that the neutral protease was not responsible of the chitosanolytic activity in the extract, whilst it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (towards human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.

Registro de acceso restringido Este recurso no está disponible en acceso abierto por política de la editorial. No obstante, se puede acceder al texto completo desde la Universitat Jaume I o si el usuario cuenta con suscripción. Registre d'accés restringit Aquest recurs no està disponible en accés obert per política de l'editorial. No obstant això, es pot accedir al text complet des de la Universitat Jaume I o si l'usuari compta amb subscripció. Restricted access item This item isn't open access because of publisher's policy. The full--text version is only available from Jaume I University or if the user has a running suscription to the publisher's contents.

Quadruplex structures have been identified in a plethora of organisms where they play important functions in the regulation of molecular processes, and hence have been proposed as therapeutic targets for many diseases. In this paper we report the extensive bioinformatic analysis of the SARS-CoV-2 genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for SARS-CoV-2. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, 512 potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over seventeen thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were then compared subsequently to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire viral realm. Sequences found in common with other viral species were further analyzed and characterized. Sequences with high scores unique to the SARS-CoV-2 were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates were then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable therapeutic targets for the SARS-CoV-2.

The eco-friendly synthesis of non-natural glycosides from different phenolic antioxidants was carried out using a fungal β-xylosidase to evaluate changes in their bioactivities. Xylosides from hydroquinone and catechol were successfully formed, although the best results were obtained for hydroxytyrosol, the main antioxidant from olive oil. The formation of the new products was followed by thin-layer chromatography, liquid chromatography, and mass spectrometry. The hydroxytyrosyl xyloside was analyzed in more detail, to maximize its production and evaluate the effect of glycosylation on some hydroxytyrosol properties. The synthesis was optimized up to the highest production reported for a hydroxytyrosyl glycoside. The structure of this compound was solved by two-dimensional nuclear magnetic resonance and identified as 3,4-dihydroxyphenyl-ethyl-O-β-d-xylopyranoside. Evaluation of its biological effect showed an enhancement of both its neuroprotective capacity and its ability to ameliorate intracellular levels of reactive oxygen species.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

We present G4-iM Grinder, a system for the localization, characterization and selection of potential G4s, i-Motifs and higher order structures. A robust and highly adaptable search engine identifies all structures that fit the user’s quadruplex definitions. Their biological relevance, in vitro formation probability and presence of known-to-form structures are then used as filters. The outcome is an efficient methodology that helps select the best candidates for a subsequent in vitro analysis or a macroscopic genomic quadruplex assessment. As proof of the analytical capabilities of G4-iM Grinder, the human genome was analyzed for potential G4s and i-Motifs. Many known-to-form structures were identified. New candidates were selected considering their score and appearance frequency. We also focused on locating Potential Higher Order Quadruplex Sequences (PHOQS). We developed a new methodology to predict the most probable subunits of these assemblies and applied it to a PHOQS candidate. Taking the human average density as reference, we examined the genomes of several etiological causes of disease. This first of its class comparative study found many organisms to be very dense in these potential quadruplexes. Many presented already known-to-form-G4s and i-Motifs. These findings suggest the potential quadruplexes have as therapeutic targets for these diseases that currently kill millions worldwide.