Magnetic solid nanoparticles and their counterparts: recent advances towards cancer theranostics

Cancer is currently a leading cause of death worldwide. The World Health Organization estimates an increase of 60% in the global cancer incidence in the next two decades. The inefficiency of the currently available therapies has prompted an urgent effort to develop new strategies that enable early diagnosis and improve response to treatment. Nanomedicine formulations can improve the pharmacokinetics and pharmacodynamics of conventional therapies and result in optimized cancer treatments. In particular, theranostic formulations aim at addressing the high heterogeneity of tumors and metastases by integrating imaging properties that enable a non-invasive and quantitative assessment of tumor targeting efficiency, drug delivery, and eventually the monitoring of the response to treatment. However, in order to exploit their full potential, the promising results observed in preclinical stages need to achieve clinical translation. Despite the significant number of available functionalization strategies, targeting efficiency is currently one of the major limitations of advanced nanomedicines in the oncology area, highlighting the need for more efficient nanoformulation designs that provide them with selectivity for precise cancer types and tumoral tissue. Under this current need, this review provides an overview of the strategies currently applied in the cancer theranostics field using magnetic nanoparticles (MNPs) and solid lipid nanoparticles (SLNs), where both nanocarriers have recently entered the clinical trials stage. The integration of these formulations into magnetic solid lipid nanoparticles—with different composition and phenotypic activity—constitutes a new generation of theranostic nanomedicines with great potential for the selective, controlled, and safe delivery of chemotherapy.This research was funded by the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia—FCT) and the European Regional Development Fund (ERDF) through NORTE 2020 (2014–2020 North Portugal Regional Operational Program) under the project NORTE-01-0145-FEDER-031142 “Local specific treatment of triple-negative-breast-cancer through externally triggered target-less drug carriers (MagtargetON)”, and by 2014–2020 INTERREG Cooperation Programme Spain–Portugal (POCTEP) through the project 0624_2IQBIONEURO_6_E

G-Quadruplex DNA as a Target in Pathogenic Bacteria:Efficacy of an Extended Naphthalene Diimide Ligand and Its Mode of Action

[Image: see text] Guanidine DNA quadruplex (G4-DNA) structures convey a distinctive layer of epigenetic information that is critical for regulating key biological activities and processes as transcription, replication, and repair in living cells. The information regarding their role and use as therapeutic drug targets in bacteria is still scarce. Here, we tested the biological activity of a G4-DNA ligand library, based on the naphthalene diimide (NDI) pharmacophore, against both Gram-positive and Gram-negative bacteria. For the best compound identified, NDI-10, a different action mechanism was described for Gram-positive or negative bacteria. This asymmetric activity profile could be related to the different prevalence of putative G4-DNA structures in each group, the influence that they can exert on gene expression, and the different roles of the G4 structures in these bacteria, which seem to promote transcription in Gram-positive bacteria and repress transcription in Gram-negatives

THIOSUGARS AS VECTORS FOR G-QUADRUPLEX LIGANDS: ANTICANCER AND ANTIPARASITIC DRUGS.

Congresos y Conferencias: Comunicación de Congreso - Conferencia invitada

Chemically Tuning Resveratrol for the Effective Killing of Gram-Positive Pathogens

In the era of antimicrobial resistance, the identification of new compounds with strong antimicrobial activity and the development of alternative therapies to fight drug-resistant bacteria are urgently needed. Here, we have used resveratrol, a safe and well-known plant-derived stilbene with poor antimicrobial properties, as a scaffold to design several new families of antimicrobials by adding different chemical entities at specific positions. We have characterized the mode of action of the most active compounds prepared and have examined their synergistic antibacterial activity in combination with traditional antibiotics. Some alkyl- and silyl-resveratrol derivatives show bactericidal activity against Gram-positive bacteria in the same low micromolar range of traditional antibiotics, with an original mechanism of action that combines membrane permeability activity with ionophore-related activities. No cross-resistance or antagonistic effect was observed with traditional antibiotics. Synergism was observed for some specific general-use antibiotics, such as aminoglycosides and cationic antimicrobial peptide antibiotics. No hemolytic activity was observed at the active concentrations or above, although some low toxicity against an MRC-5 cell line was noted

Glycosyl conjugation to G-quadruplex ligands as a strategy to improve their antitumor and antiparasitic efficacy.

Congresos y Conferencias: Comunicación de Congreso - Conferencia invitada

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase

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.This research was funded by the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601-C3-1-R and BIO2016-76601-C3-3-R).Peer reviewe

Glycosyl and alkyl modifications on potential therapeutic drugs targeting parasitic, cancer and neurodegenerative diseases

La presente tesis doctoral está centrada en el desarrollo de conjugados de potenciales fármacos con carbohidratos y cadenas lipófilas. Estos compuestos que se han considerado “Lead” a optimizar se basan en ligandos de ADN en G-quadruplex (G4) (véase la siguiente ilustración) y en tres fenoles naturales, tirosol (Tyr), hidroxitirosol (HT) y resveratrol (RES). En primer lugar, hemos diseñado y sintetizado conjugados de azúcares y ligandos de G4 como posibles agentes anticancerígenos y antiparasitarios. La idea es obtener compuestos con mejores propiedades farmacológicas, mejor afinidad hacia sus dianas terapéuticas y mejor selectividad hacia células tumorales y parásitos protozoarios. La diana terapéutica son los ADN en G-quadruplex (G4), estructuras secundarias que se forman en secuencias del ADN y ARN ricas en guaninas vinculadas a la regulación de importantes procesos biológicos, incluidos la replicación y la transcripción de determinados genes. Se ha sintetizado la nueva familia de ligandos de G-quadruplex basadas en las naftalendiimidas (NDI) en colaboración con el Prof. Mauro Freccero (Universidad de Pavia, Italia). La preparación de los conjugados se realizó por medio de la cicloadición 1,4- de Huisgen (click chemistry) usando como precursores azidoazúcares y un NDI modificado con un alquino. Los carb-NDI se unen fuertemente a sus dianas y muestran selectividad frente a dúplex de ADN. La presencia del carbohidrato reduce ligeramente la afinidad por las dianas si se compara con el control aglicona-NDI pero aún así también son buenos ligandos de las secuencias mayoritarias de Trypanosoma brucei y Leishmania major, encontradas mediante una búsqueda in silico de la incidencia y función biológica de G4 en sus respectivos genomas, y de la secuencia del telomero humano. Se han realizado estudios de entrada celular de los ligandos carb-NDI mediante citometría de flujo y espectroscopía de fluorescencia en diferentes líneas cancerígenas y no tumorales. Pudimos observar diferencias importantes entre el control sin azúcar, que se localizan en el núcleo de manera casi instantánea independientemente de la temperatura, y los conjugados carb-NDI, que también se acumulan en el núcleo pero más lentamente. En este último casi su entrada depende de la temperatura, tiempo de incubación y del tipo de unión del carbohidrato. Empleando incubaciones con diferentes inhibidores de GLUT (transportadores de glucosa), se observó que para la entrada de los carb-NDI en las células se producía, al menos parcialmente, mediante GLUT4. Se observó una correlación entre entrada celular y viabilidad de las células tumorales tras incubación con los derivados NDI. El control aglicona-NDI que entra más rápidamente es el más citotóxico, pero no el más selectivo hacia las líneas cancerígenas. Los carb-NDI mostraron actividad antiproliferativa en el rango submicromolar y son potenciales candidatos a ser ensayados in vivo. La actividad antiparasitaria de los carb-NDI fue destacable en los diferentes parásitos, en especial en Trypanosoma brucei y Leishmania major donde se observaron algunas diferencias dependiendo del carbohidrato y del tipo de unión. En todos los casos, la fluorescencia inherente en los compuestos permitió localizar los conjugados principalmente en el núcleo y el kinetoplasto usando microscopía confocal. Los ensayos de toxicidad in vivo en un modelo de pez cebra demostraron que los compuestos con carbohidrato eran menos tóxicos que el control aglicona-NDI. En segundo lugar, se sintetizaron diferentes derivados glicosilados y alquilados de hidroxitirosol, tirosol y resveratrol. Los derivados de HT y Tyr fueron evaluados como potenciales agentes antiparasitarios mostrando una interesante actividad y selectividad especialmente frente a Trypanosoma brucei. El estudio de la relación estructura-actividad de los compuestos determinó que la longitud óptima de la cadena alifática es de entre 10 y 12 carbonos. Por otro lado, la forma de unión de la cadena alifática al HT parece menos importante, al menos según los resultados obtenidos en el modelo in vitro utilizado. Los derivados alquilados y glicosilados de RES fueron evaluados como potenciales agentes neuroprotectores y anti-inflamatorios. Algunos de estos compuestos demostraron una actividad mejor que el propio RES in vitro. Esta mejora también fue observada en dos modelos in vivo, uno de pez cebra y otro murino. El compuesto 3-O-(6’-O-octanoil)--D-glucopiranosido resveratrol (véase debajo) demostró la mejor actividad neuroprotectora y anti-inflamatoria, señalándolo como un potencial fármaco para enfermedades neurodegenerativas.Tesis Univ. Granada.Programa Oficial de Doctorado en FarmaciaWe gratefully acknowledge to Junta de Andalucía (Grant No. FQM-7316 (JCM), BIO-1786 (JMPV), CTS-7282 (FG)) and Spanish Ministry of Economy and Competitiveness (Grant No. SAF2011-28215 (JMPV) and SAF2012-34267 (FG)) for financial support

Exploring G and C-quadruplex structures as potential targets against the severe acute respiratory syndrome coronavirus 2

In this paper we report the analysis of the 2019-nCoV 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 2019-nCoV. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, hundreds of 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 three thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were compared sequentially to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire realm. Sequences found in common with other species were further analyzed and characterized. Sequences with high scores unique to the 2019-nCoV were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates was then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable theranostic targets against the 2019-nCoV.This work was supported by the 2014-2020 North Portugal Regional Operational Program (NORTE 2020) and the European Regional Development Fund (ERDF) under Grant NORTE-01- 0145-FEDER-000019, by the Fundação para a Ciência e a Tecnoloxía (FCT), ERDF and NORTE 2020 through the Grant NORTE-01-0145-FEDER-031142 (Local specific treatment of triplenegative-breast-cancer through externally triggered target-less drug carriers – MagtargetON –), and by the 2014-2020 INTERREG Cooperation Programme Spain–Portugal (POCTEP) through the project 0624_2IQBIONEURO_6_E. C. G. also acknowledges support from the Spanish Ministry of Science, Innovation and Universities (MCIU) under project BFU2017-89707-P.N

Combining experimental data with statistical methods to evaluate hydrolyzed reactive dye removal by α-Fe2O3 in a cellulose-based membrane

Water contaminated with toxic dyes poses serious problems for human health and environmental ecosystems. Unfixed reactive dyes and their hydrolyzed form are soluble in water, thus, their removal is particularly challenging. Among the different methodologies, adsorption is probably the most common since it is easy to handle and has a low cost. Here, the removal by adsorption of hydrolyzed Reactive Black 5 (hydRB5) from a model wastewater through cellulose acetate/hematite membranes (CA/α-Fe2O3), designated as M1, M2 and M3, was performed. The pristine cellulose acetate membrane (CA) was designated as M0. Toward understanding the adsorption mechanism of hydRB5 on membranes, the rate of adsorption and maximum value of the adsorption capacity were evaluated using kinetic and isothermal studies, respectively. The results showed that the adsorption mechanism follows pseudo-first-order kinetics, and data are best fitted by the Langmuir isotherm method with a maximum adsorption capacity of 105.26 mg g−1 in pH~7. Furthermore, these membranes can be also regenerated by washing with NaOH and NaCl solutions, and the regeneration efficiency remains effective over five cycles. To complete the work, two statistical models were applied, an Analysis of Variance (ANOVA) and a Response Surface Methodology (RSM). The optimum value found is located in the usable region, and the experimental validation shows good agreement between the predicted optimum values and the experimental data. These composite membranes are also good candidates for the adsorption of other pollutants, even at industrial scale, due to their effective regeneration process and low production costs.This work was funded by NORTE2020, funding reference NORTE-01-0145-FEDER-000015, within the Project TSSiPRO-Technologies for Sustainable and Smart Innovative Products, and also by national funds through FCT—Foundation for Science and Technology within the scope of the PROJECT UID/CTM/00264/2013

Potential G-quadruplexes and i-Motifs in the SARS-CoV-2.

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