Home Publications

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

Grijalvo, S., Alagia, A., Jorge, A.F., Eritja, R.

Genes, 9 (2), art. no. 74, 2018

Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.


Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex

Mir, J.F., Zagmutt, S., Lichtenstein, M.P., García-Villoria, J., Weber, M., Gracia, A., Fabriàs, G., Casas, J., López, M., Casals, N., Ribes, A., Suñol, C., Herrero, L., Serra, D.

Molecular Neurobiology, 2018

 Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and ?-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and a-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.


A lamellar body mimetic system for the treatment of oxazolone-induced atopic dermatitis in hairless mice

Moner, V., Fernández, E., Calpena, A.C., Garcia-Herrera, A., Cócera, M., López, O.

Journal of Dermatological Science, 2018

 Background Atopic dermatitis is a common skin disease characterized by a Th2 cell-dominant inflammatory infiltrate, elevated serum IgE levels and impaired epidermal barrier function. It is associated to abnormal epidermal lamellar body secretion, producing alteration in lipid composition and extracellular lamellar membrane organization. Objectives The oxazolone-induced atopic dermatitis in hairless mice was used to evaluate in vivo the effect of the application of a lipid system that mimics the morphology, structure and composition of epidermal lamellar bodies. Methods The skin barrier function was evaluated measuring TEWL and skin hydration in vivo. Inflammation was assessed by analysis of serum IgE levels and histological analysis. The microstructure of the intercellular lipid region was also evaluated before and after treatment. Results The skin condition was improved after 10 days of treatment indicated by decreased TEWL, decreased serum IgE levels, reduced epidermal thickness and reduced lymphocyte-dominated infiltrate. However, the treatment did no improve skin hydration. Conclusions The treatment with this lipid system seems to improve the skin condition by reinforcing the barrier function and reducing the skin inflammation. Therefore, the present study provides evidence that this lipid system combining appropriate lipid composition and morphology could be of interest for the development of future treatments for atopic dermatitis.


Hyaluronan based materials with catanionic sugar-derived surfactants as drug delivery systems

Roig, F., Blanzat, M., Solans, C., Esquena, J., García-Celma, M.J.

 Colloids and Surfaces B: Biointerfaces, 164, 218-223, 2018

 In the present work novel drug delivery systems consisting in highly porous Hyaluronan foams for the administration of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, have been obtained. A sugar-derived surfactant associated with ketoprofen was prepared and incorporated into the porous hyaluronan materials. The association between a lactose derived surfactant, Lhyd12, and ketoprofen was obtained by acid-base reaction and its physicochemical properties were studied. Tensiometric and dynamic light scattering (DLS) determinations showed the formation of catanionic surfactant aggregates, Lhyd12/ketoprofen, in aqueous solution. Furthermore, the catanionic surfactants allowed greater solubilisation of ketoprofen. Hyaluronan porous materials were developed using butanediol diglycidyl ether as crosslinking agent. The profile release of Lhyd12/ketoprofen from hyaluronan based materials shows differences as a function of the aggregation state of catanionic surfactant.


Electrochemical and AFM characterization of G-quadruplex electrochemical biosensors and applications

Chiorcea-Paquim, A.-M., Eritja, R., Oliveira-Brett, A.M.

 Journal of Nucleic Acids, 5307106, 2018

 Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited.


Compression of multidimensional NMR spectra allows a faster and more accurate analysis of complex samples

 Francesc Puig-Castellví,  Yolanda Pérez,  Benjamín Piña,  Romà Tauler  and  Ignacio Alfonso

 Chem. Commun., 54, 3090-3093, 2018

 We propose an approach to efficiently compress and denoise multidimensional NMR spectral data, improving their corresponding storage, handling, and analysis. This method has been tested with 2D homonuclear, 2D and 3D heteronuclear, and 2D phase-sensitive NMR spectral data and shown to be especially powerful for 2D NMR metabolomics studies.


Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside increases human immunodeficiency virus replication and viral outgrowth efficacy in vitro

Olvera, A., Martinez, J.P., Casadellà, M., Llano, A., Rosás, M., Mothe, B., Ruiz-Riol, M., Arsequell, G., Valencia, G., Noguera-Julian, M., Paredes, R., Meyerhans, A., Brander, C.

 Front. Immunol., 26 January 2018

 Glycosylation of host and viral proteins is an important posttranslational modification needed to ensure correct function of glycoproteins. For this reason, we asked whether inhibition of O-glycosylation during human immunodeficiency virus (HIV) in vitro replication could affect HIV infectivity and replication rates. We used benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN), a compound that has been widely used to inhibit O-glycosylation in several cell lines. Pretreatment and culture of PHA-blast target cells with BAGN increased the percentage of HIV-infected cells (7.6-fold, p = 0.0115), the per-cell amount of HIV p24 protein (1.3-fold, p = 0.2475), and the viral particles in culture supernatants (7.1-fold, p = 0.0029) compared to BAGN-free cultures. Initiating infection with virus previously grown in the presence of BAGN further increased percentage of infected cells (30-fold, p < 0.0001), intracellular p24 (1.5-fold, p = 0.0433), and secreted viral particles (74-fold, p < 0.0001). BAGN-treated target cells showed less CD25 and CCR5 expression, but increased HLA-DR surface expression, which positively correlated with the number of infected cells. Importantly, BAGN improved viral outgrowth kinetics in 66% of the samples tested, including samples from HIV controllers and subjects in whom no virus could be expanded in the absence of BAGN. Sequencing of the isolated virus indicated no skewing of viral quasi-species populations when compared to BAGN-free culture conditions. BAGN also increased virus production in the ACH2 latency model when used together with latency-reversing agents. Taken together, our results identify BAGN treatment as a simple strategy to improve viral outgrowth in vitro and may provide novel insights into host restriction mechanisms and O-glycosylation-related therapeutic targets for HIV control strategies.


Nucleophile Promiscuity of Engineered Class II Pyruvate Aldolase YfaU from E. Coli

Karel Hernández, Jesúls Joglar, Jordi Bujons, Teodor Parella, and Pere Clapés

 Angew.Chem.Int.Ed., 2018

 Pyruvate-dependent aldolases exhibit a stringent selectivity for pyruvate, limiting application of their synthetic potential, which is a drawback shared with other existing aldolases. Structure-guided rational protein engineering rendered a 2-keto-3-deoxy-l-rhamnonate aldolase variant, fused with a maltose-binding protein (MBP-YfaU W23V/L216A), capable of efficiently converting larger pyruvate analogues, for example, those with linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N-Cbz-alaninal (Cbz=benzyloxycarbonyl) and N-Cbz-prolinal electrophiles gave access to chiral building blocks, for example, derivatives of (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid (68?%, d.r. 90:10) and the enantiomer of dolaproine (33?%, d.r. 94:6) as well as a collection of unprecedented a-amino acid derivatives of the proline and pyrrolizidine type. Conversions varied between 6–93?% and diastereomeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components.


Release of small bioactive molecules from physical gels

Mayr, J.; Saldías, C.; Díaz, D. D.

Chem. Soc. Rev. 2018

 Pharmaceutical drugs with low water solubility have always received great attention within the scientific community. The reduced bioavailability and the need of frequent administrations have motivated the investigation of new drug delivery systems. Within this context, drug carriers that release their payload in a sustained way and hence reduce the administration rate are highly demanded. One interesting strategy to meet these requirements is the entrapment of the drugs into gels. So far, the most investigated materials for such drug-loaded gels are derived from polymers and based on covalent linkages. However, over the last decade the use of physical (or supramolecular) gels derived from low molecular weight compounds has experienced strong growth in this field, mainly due to important properties such as injectability, stimuli responsiveness and ease of synthesis. This review summarizes the use of supramolecular gels for the encapsulation and controlled release of small therapeutic molecules.


Reactivity of hydropersulfides toward the hydroxyl radical unraveled: disulfide bond cleavage, hydrogen atom transfer, and proton-coupled electron transfer

Anglada, J.M., Crehuet, R., Adhikari, S., Francisco, J.S., Xia, Y.

Physical Chemistry Chemical Physics, 20 (7), 4793-4804, 2018

 Hydropersulfides (RSSH) are highly reactive as nucleophiles and hydrogen atom transfer reagents. These chemical properties are believed to be key for them to act as antioxidants in cells. The reaction involving the radical species and the disulfide bond (S–S) in RSSH, a known redox-active group, however, has been scarcely studied, resulting in an incomplete understanding of the chemical nature of RSSH. We have performed a high-level theoretical investigation on the reactions of the hydroxyl radical (?OH) toward a set of RSSH (R = –H, –CH3, –NH2, –C(O)OH, –CN, and –NO2). The results show that S–S cleavage and H-atom abstraction are the two competing channels. The electron inductive effect of R induces selective ?OH substitution at one sulfur atom upon S–S cleavage, forming RSOH and ?SH for the electron donating groups (EDGs), whereas producing HSOH and ?SR for the electron withdrawing groups (EWGs). The H-Atom abstraction by ?OH follows a classical hydrogen atom transfer (hat) mechanism, producing RSS? and H2O. Surprisingly, a proton-coupled electron transfer (pcet) process also occurs for R being an EDG. Although for RSSH having EWGs hat is the leading channel, S–S cleavage can be competitive or even dominant for the EDGs. The overall reactivity of RSSH toward ?OH attack is greatly enhanced with the presence of an EDG, with CH3SSH being the most reactive species found in this study (overall rate constant: 4.55 × 1012 M-1 s-1). Our results highlight the complexity in RSSH reaction chemistry, the extent of which is closely modulated by the inductive effect of the substituents in the case of the oxidation by hydroxyl radicals.


Page 4 of 94