Home Publications

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.


Exploring PAZ/3'-overhang interaction to improve siRNA specificity. A combined experimental and modeling study

Alagia, A., Jorge, A.F., Aviñó, A., Cova, T.F.G.G., Crehuet, R., Grijalvo, S., Pais, A.A.C.C., Eritja, R.

Chemical Science, 9 (8), pp. 2074-2086. 2018

 The understanding of the dynamical and mechanistic aspects that lie behind siRNA-based gene regulation is a requisite to boost the performance of siRNA therapeutics. A systematic experimental and computational study on the 3'-overhang structural requirements for the design of more specific and potent siRNA molecules was carried out using nucleotide analogues differing in structural parameters, such as sugar constraint, lack of nucleobase, distance between the phosphodiester backbone and nucleobase, enantioselectivity, and steric hindrance. The results established a set of rules governing the siRNA-mediated silencing, indicating that the thermodynamic stability of the 5'-end is a crucial determinant for antisense-mediated silencing but is not sufficient to avoid sense-mediated silencing. Both theoretical and experimental approaches consistently evidence the existence of a direct connection between the PAZ/3'-overhang binding affinity and siRNA’s potency and specificity. An overall description of the systems is thus achieved by atomistic simulations and free energy calculations that allow us to propose a robust and self-contained procedure for studying the factors implied in PAZ/3'-overhang siRNA interactions. A higher RNAi activity is associated with a moderate-to-strong PAZ/3'-overhang binding. Contrarily, lower binding energies compromise siRNA potency, increase specificity, and favor siRNA downregulation by Ago2-independent mechanisms. This work provides in-depth details for the design of powerful and safe synthetic nucleotide analogues for substitution at the 3'-overhang, enabling some of the intrinsic siRNA disadvantages to be overcome.


Effect of Surface Chemistry and Associated Protein Corona on the Long-Term Biodegradation of Iron Oxide Nanoparticles In Vivo

Stepien, G., Moros, M., Pérez-Hernández, M., Monge, M., Gutiérrez, L., Fratila, R.M., Las Heras, M.D., Menao Guillén, S., Puente Lanzarote, J.J., Solans, C., Pardo, J., De La Fuente, J.M.

ACS Applied Materials and Interfaces, 10 (5), pp. 4548-4560. 2018

 The protein corona formed on the surface of a nanoparticle in a biological medium determines its behavior in vivo. Herein, iron oxide nanoparticles containing the same core and shell, but bearing two different surface coatings, either glucose or poly(ethylene glycol), were evaluated. The nanoparticles’ protein adsorption, in vitro degradation, and in vivo biodistribution and biotransformation over four months were investigated. Although both types of nanoparticles bound similar amounts of proteins in vitro, the differences in the protein corona composition correlated to the nanoparticles biodistribution in vivo. Interestingly, in vitro degradation studies demonstrated faster degradation for nanoparticles functionalized with glucose, whereas the in vivo results were opposite with accelerated biodegradation and clearance of the nanoparticles functionalized with poly(ethylene glycol). Therefore, the variation in the degradation rate observed in vivo could be related not only to the molecules attached to the surface, but also with the associated protein corona, as the key role of the adsorbed proteins on the magnetic core degradation has been demonstrated in vitro.


Oleic acid chlorohydrin, a new early biomarker for the prediction of acute pancreatitis severity in humans

de-Madaria, E., Molero, X., Bonjoch, L., Casas, J., Cárdenas-Jaén, K., Montenegro, A., Closa, D.

Annals of Intensive Care, 2018, 8:1

 Background The early prediction of the severity of acute pancreatitis still represents a challenge for clinicians. Experimental studies have revealed the generation of specific halogenated lipids, in particular oleic acid chlorohydrin, in the early stages of acute pancreatitis. We hypothesized that the levels of circulating oleic acid chlorohydrin might be a useful early prognostic biomarker in acute pancreatitis in humans. Methods In a prospective, multicenter cohort study, plasma samples collected within 24 h after presentation in the emergency room from 59 patients with acute pancreatitis and from 9 healthy subjects were assessed for oleic acid chlorohydrin levels. Results Pancreatitis was mild in 30 patients, moderately severe in 16 and severe in 13. Oleic acid chlorohydrin levels within 24 h after presentation were significantly higher in patients that later progressed to moderate and severe acute pancreatitis. Using 7.49 nM as the cutoff point, oleic acid chlorohydrin distinguished mild from moderately severe-to-severe pancreatitis with high sensitivity/specificity (96.6/90.0%) and positive/negative predictive values (90.3/96.4%). Using 32.40 nM as the cutoff value sensitivity, specificity, positive and negative predictive values were all 100% for severe acute pancreatitis. It was found to be a better prognostic marker than BISAP score, hematocrit at 48 h, SIRS at admission, persistent SIRS or C-reactive protein at 48 h. Conclusions Oleic acid chlorohydrin concentration in plasma is elevated in patients with acute pancreatitis on admission and correlates with a high degree with the final severity of the disease, indicating that it has potential to serve as an early prognostic marker for acute pancreatitis severity.


Stability, biocompatibility and antioxidant activity of PEG-modified liposomes containing resveratrol

Caddeo, C., Pucci, L., Gabriele, M., Carbone, C., Fernàndez-Busquets, X., Valenti, D., Pons, R., Vassallo, A., Fadda, A.M., Manconi, M.

International Journal of Pharmaceutics, 538 (1-2), pp. 40-47., 2018


The present investigation reports the development of PEG-modified liposomes for the delivery of naturally occurring resveratrol. PEG-modified liposomes were prepared by direct sonication of the phospholipid aqueous dispersion, in the presence of two PEG-surfactants. Small, spherical, unilamellar vesicles were produced, as demonstrated by light scattering, cryo-TEM, and SAXS. The aging of the vesicles was assessed by using the Turbiscan® technology, and their physical stability was evaluated in vitro in simulated body fluids, results showing that the key features of the liposomes were preserved. The biocompatibility of the formulations was demonstrated in an ex vivo model of hemolysis in human erythrocytes. Further, the incorporation of resveratrol in PEG-modified liposomes did not affect its intrinsic antioxidant activity, as DPPH radical was almost completely inhibited, and the vesicles were also able to ensure an optimal protection against oxidative stress in an ex vivo human erythrocytes-based model. Therefore, the proposed PEG-modified liposomes, which were prepared by a simple and reliable method, represent an interesting approach to safely deliver resveratrol, ensuring the preservation of the carrier structural integrity in the biological fluids, and the antioxidant efficacy of the polyphenol to be exploited against oxidative stress associated with cancer.


Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration

Kotronoulas, A., Gomez-Gómez, A., Fabregat, A., Segura, J., Yang, S.h, Xing, Y., Moutian, W., Marcos, J., Joglar, J., Ventura, R., Pozo, O.J.

Drug Testing and Analysis, 2017

In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1-cyclopentenoylglycine (1-CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra-individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1-CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration

Kotronoulas, A., Gomez-Gómez, A., Fabregat, A., Segura, J., Yang, S.h, Xing, Y., Moutian, W., Marcos, J., Joglar, J., Ventura, R., Pozo, O.J.

Drug Testing and Analysis, 2017

Although the introduction by the World Anti-Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L-T/E) and medium basal (M-T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L-T/E and 6 M-T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra-individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra-individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra-individual variability, the DWs reached by glucuronoconjugates (2–3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.

Page 4 of 93