Browsing by Author "Universidad San Pablo-CEU. Departamento de Ciencias Farmacéuticas y de la Salud"

5-fluorouracil (FU) is a pyrimidine analogue extensively used in cancer chemotherapy. FU can be metabolized into 5-fluorouridine-triphosphate, which can be used as substrate for viral RNA-dependent RNA polymerases. This results in the incorporation of mutations into viral RNA. Accumulation of mutations may lead to loss of virus infectivity, in a process known as lethal mutagenesis. RNA virus pathogens are particularly difficult to control because they are highly mutable, and mutants resistant to antiviral agents are readily selected. Here, we review the basic principles of lethal mutagenesis as an antiviral approach, and the participation of FU in its development. Recent studies with foot-and-mouth disease virus indicate that FU can act both as an inhibitor and as a mutagen during foot-and-mouth disease virus replication. This dual activity renders FU an adequate drug for lethal mutagenesis. We suggest that structural and biochemical studies can contribute to the lead to new design of base or nucleoside analogues targeted specifically to viral polymerases.

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

Objetivo: cuantificar las ingestas dietéticas de los micronutrientes implicados en el ciclo metilación-metionina (colina, betaína, folatos, vitaminas (B6 y B12) en una muestra representativa de mujeres gestantes residentes en España; determinar la adecuación a las recomendaciones, y analizar sus principales fuentes alimentarias. Material y métodos: la determinación de la ingesta media se realizó a partir de los datos de consumo de los alimentos recogidos en la “Encuesta Nacional de Alimentación en población adulta, mayores y embarazadas” (ENALIA-2) (n = 133). Para el cálculo del aporte de folatos y de vitaminas B6 y B12 se emplearon los datos de composición nutricional recogidos en las “Tablas de Composición de Alimentos en España”, mientras que para la colina y la betaína, nutrientes no incluidos en las bases de datos de composición de alimentos en Europa, se empleó la “Base de Datos Nacional de Nutrientes para Referencia Estándar del Departamento de Agricultura de los Estados Unidos” (USDA). La adecuación de la ingesta se estimó de acuerdo con las recomendaciones de las principales guías españolas, europeas y estadounidenses. Resultados: las ingestas medias diarias observadas fueron de 271,1 mg/día de colina; 142,5 mg/día de betaína; 182,8 μg/día de folatos; 1,4 mg/día de vitamina B6; y 4,5 μg/día de vitamina B12. Los niveles de adecuación a las recomendaciones resultaron insuficientes para la colina (< 60,2 %) y los folatos (< 30,5 %); cercanos a la adecuación para la vitamina B6 (> 71,6 %); y plenamente adecuados únicamente en el caso de la vitamina B12 (> 101,1 %). No resulta posible extraer ninguna conclusión con respecto al aporte de betaína al no existir recomendaciones establecidas. Las principales fuentes alimentarias fueron: alimentos de origen animal para la colina y la vitamina B12 (71,8 % y 97,4 %, respectivamente); cereales y derivados para la betaína (85,3 %); verduras y hortalizas (27,5 %) junto a cereales y derivados (18,6 %) para los folatos; y carnes y derivados (26,6 %), seguidos de verduras y hortalizas (17,9 %) para la vitamina B6. Conclusiones: los resultados obtenidos son indicativos de la necesidad de mejorar la ingesta y el estado nutricional de estos componentes de gran interés para la salud de la mujer embarazada. Como consecuencia del grado de adecuación observado, parece necesario y urgente el empleo no solo de estrategias para mejorar la dieta y el uso de alimentos fortificados, sino también de suplementos nutricionales de manera personalizada.

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.

Designer cells for a synthetic cascade reaction harnessing selective redox reactions were devised, featuring two successive regioselective P450-catalyzed CH-activating oxidations of 1-cyclohexene carboxylic acid methyl ester followed by stereoselective olefin-reduction catalysed by (R)- or (S)-selective mutants of an enoate reductase.

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fuorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fuorescent dye, which permits the specifc quantifcation and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fuorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identifcation of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by diferent RdRps, illustrated here using purifed RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fuorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.

The achiral cyclohexene derivative dimethyl cis-1,2,3,6-tetrahydrophthalate has been subjected to oxidation catalyzed by cytochrome P450 monooxygenase P450-BM3, leading to diastereoselective epoxidation rather than oxidative hydroxylation. This reaction occurs with 94% diastereoselectivity in favor of the anti-epoxide, in contrast to m-CPBA which delivers unselectively a 70:30 mixture of anti/syn diastereomers. The experimental results are nicely explained on a molecular level by docking experiments and molecular dynamics computations.

Growing evidence confirms choline as a critical perinatal nutrient. However, intake levels of choline and betaine among the Spanish fertile population remain unknown. Given their role in one-carbon metabolism with potential epigenetic effects, the aim of the present study was to evaluate the dietary intakes, their adequacy to existing guidelines and the main food sources together with other micronutrients involved in the methylation-methionine cycle (vitamin B6, folates and vitamin B12) in women of childbearing age. The ANIBES study, a cross-sectional study of a representative sample of women of childbearing age (18–45 years, n = 641) resident in Spain, was used. The sample was divided into younger women (18–30 years, n = 251) and older women (31–45 years, n = 390). Dietary intake was assessed by a three-day dietary record by using a tablet device. Total median intakes for the total sample were 303.9 mg/d for choline; 122.6 mg/d for betaine; 1.3 mg/d for vitamin B6; 140.8 g/d for folates, and 3.8 g/d for vitamin B12. The older subgroup showed significantly higher choline (p < 0.05), betaine (p < 0.001) and folates (p < 0.05) intakes than younger women. Main food sources for the whole sample were meat and meat products for choline (28.3%), vitamin B6 (25.7%) and vitamin B12 (22.8%); cereals and derivatives (79.9%) for betaine; vegetables (20.0%) for folates. Overall intake adequacy was only observed for vitamin B12, with a very limited number of participants showing adequate intakes for all the other micronutrients. These results illustrate there is a relevant need to raise awareness about optimizing the status of the methionine cycle-related vitamins and cofactors in this potentially vulnerable population.

Iterative saturation mutagenesis (ISM) in combination with reduced amino acid alphabets has been shown to be an efficient method for directed evolution. In order to minimize the screening effort, the number of residues in a given randomization site has thus far been restricted to two or three; this prevents oversampling from reaching astronomical numbers when 95 % library coverage is aimed for. In this study, ISM is applied for the first time by using randomization sites composed of five amino acid positions. The use of just two such sites (A and B) results in two different ISM pathways, A→B and B→A. A severely reduced amino acid alphabet (only five members) was employed for the building blocks-a minimal set of structurally representative amino acids. The Baeyer-Villiger monooxygenase PAMO was chosen as the enzyme for this proof-of-principle study. The test system employed tuning of activity and diastereoselectivity in the oxidation of 4-(bromomethylidene)cyclohexanone, which is not accepted by wild-type PAMO. Although only 8-9 % library coverage was ensured (as calculated by traditional statistics), notable activity and 99 % diastereoselectivity were obtained, thus indicating that such an ISM strategy is viable in protein engineering.

tDirected evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asym-metric transformations in organic chemistry and biotechnology. Almost all of the numerous examplesreported in the literature utilize high-throughput screening systems based on suitable analytical tech-niques. Since the screening step is the bottleneck of the overall procedure, researchers have consideredthe use of genetic selection systems as an alternative to screening. In principle, selection would be themost elegant and efficient approach because it is based on growth advantage of host cells harboring ste-reoselective mutants, but devising such selection systems is very challenging. They must be designed sothat the host organism profits from the presence of an enantioselective variant. Progress in this intrigu-ing research area is summarized in this review, which also includes some examples of display systemsdesigned for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although thecombination of display systems and FACS is a powerful approach, we also envision innovative ideascombining metabolic engineering and genetic selection systems with protein directed evolution for thedevelopment of highly selective and efficient biocatalysts.

Biological drugs targeting tumour necrosis factor are effective for psoriasis. However, 30-50% of patients do not respond to these drugs and may even develop paradoxical psoriasiform reactions. This study search-ed for DNA copy number variations that could predict anti-tumour necrotic factor drug response or the appearance of anti-tumour necrotic factor induced psoriasiform reactions. Peripheral blood samples were collected from 70 patients with anti-tumour necrotic factor drug-treated moderate-to-severe plaque psoriasis. Samples were analysed with an Illumina 450K methylation microarray. Copy number variations were obtained from raw methylation data using conumee and Chip Analysis Methylation Pipeline (ChAMP) R packages. One copy number variation was found, harbouring one gene (CPM) that was significantly associated with adalimumab response (Bonferroni-adjusted p-value < 0.05). Moreover, one copy number variation was identified harbouring 3 genes (ARNT2, LOC101929586 and MIR5572) related to the development of paradoxical psoriasiform reactions. In conclusion, this study has identified DNA copy number variations that could be good candidate markers to predict response to adalimumab and the development of anti-tumour necrotic factor paradoxical psoriasiform reactions.

Overweight and obesity amongst childhood are currently global health issues. However, this is the best stage of life to prevent diseases and to promote healthy habits. In our study, we evaluate the effectiveness of the THAO Salud Infantil, a community-based intervention program, by means of a cross-sectional study carried out from 2009 to 2019 surveying children aged 3 to 12 years old (n = 27,686). During the study timeframe, overweight and obesity prevalence, according to both the International Obesity Task Force and Orbegozo Foundation criteria, showed a downward trend. Differences in the anthropometric variables were observed from the beginning to the end of the study, mainly in girls. Analysis of the influence of the socioeconomic status revealed that children from families with lower incomes are in greater risk of suffering from overweight and obesity and showed lower effectiveness of the actions proposed by the program. The overall results of the study confirmed the effectiveness of community-based interventions in terms of childhood overweight/obesity prevention.

We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.

While anti-TNF therapies are effective against psoriasis, 30%–50% of patients do not show an adequate response to these rugs. Different candidate-gene pharmacogenetics studies have identified single nucleotide polymorphisms that may predict anti-TNF drugs response in psoriasis. Nevertheless, only one paper has undertaken a pharmacogenomic approach failing to find significant biomarkers of biological drug response along the whole genome. Furthermore, most of the pharmacogenetic candidate biomarkers identified previously have not been confirmed in a different cohort of patients. The objective of this study was to find biomarkers that could predict anti-TNF drugs response along the whole genome and validate biomarkers identified previously. A genome-wide association study (GWAS) was performed using the Human Omni Express-8 v1.2 Beadchips in 243 psoriasis patients treated with anti-TNF drugs. This study was multicentric and did not interfere with clinical practice. Associations between single nucleotide polymorphisms (SNP) and PASI75 (a 75% reduction with respect to baseline PASI) at 3 months were evaluated. Imputation was performed using SNPs with R2 > 0.7. There were two SNPs located in NPFFR2 that were close to the significant threshold of 5 × 10−8. These data suggest that NPFFR2 might be associated with anti-TNF drug response. However, further studies involving a larger cohort of patients are needed in order to confirm these results.

Catalytic asymmetric reduction of prochiral ketones of type 4-alkylidene cyclohexanone with formation of the corresponding axially chiral R-configurated alcohols (up to 99% ee) was achieved using alcohol dehydrogenases, whereas chiral transition-metal catalysts fail. Reversal of enantioselectivity proved to be possible by directed evolution based on saturation mutagenesis (up to 98% ee (S)). Utilization of ketone with a vinyl bromide moiety allows respective R- and S-alcohols to be exploited as key compounds in Pd-catalyzed cascade reactions.

Lethal mutagenesis is an antiviral strategy that aims to extinguish viruses as a consequence of enhanced mutation rates during virus replication. The molecular mechanisms that underlie virus extinction by mutagenic nucleoside analogues are not well understood. When mutagenic agents and antiviral inhibitors are administered sequentially or in combination, interconnected and often conflicting selective constraints can influence the fate of the virus either towards survival through selection of mutagenescape or inhibitor-escape mutants or towards extinction. Here we report a study involving the mutagenesis of foot-and-mouth disease virus (FMDV) by the nucleoside analogue ribavirin (R) and the effect of R-mediated mutagenesis on the selection of FMDV mutants resistant to the inhibitor of RNA replication, guanidine hydrochloride (GU). The results show that under comparable (and low) viral load, an inhibitory activity by GU could not substitute for an equivalent inhibitory activity by R in driving FMDV to extinction. Both the prior history of R mutagenesis and the viral population size influenced the selection of GU-escape mutants. A sufficiently low viral load allowed continued viral replication without selection of inhibitor-escape mutants, irrespective of the history of mutagenesis. These observations imply that reductions of viral load as a result of a mutagenic treatment may provide an opportunity either for immune-mediated clearing of a virus or for an alternative antiviral intervention, even if extinction is not initially achieved.

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that is being recognized as a major player in neuropsychiatric disorders such as Major Depressive Disorder (MDD). P2X7R activation is triggered by high extracellular ATP concentrations, leading to channel opening and inducing an increase in cytosolic calcium concentration ([Ca2+]c), that activates the inflammatory pathway. Those receptors are expressed not only in CNS cells but also in peripheral blood cells, where they are activated in response to inflammatory molecules such as bacterial lipopolysaccharide (LPS). LPS induced-tissue damage promotes an elevation of extracellular ATP, triggering the NRLP3-inflammasome assembly and activation that, sequentially, induces caspase-1 cleavage and IL-1β processing and secretion. In this context, we attempt to understand the role of P2X7R in [Ca2+]c homeostasis regulation, inflammasome expression and its pharmacological modulation in MDD. For this purpose, monocytes were isolated from peripheral blood of MDD patients and [Ca2+]c was monitored with the intracellular probe Fura-2. Our results point out to P2X7R as the responsible of the Ca2+ imbalance, as well as TNFα-dependent activation of caspase-1 in MDD patients. In addition, P2X7R blockade with its specific antagonist, JNJ-47965567, reduces the Ca2+ entry upon Bz-ATP exposure. Altogether, our results point that MDD patients have both, Ca2+ homeostasis alteration and an inflammatory status, which promote an independentinflammasome activation of caspase-1. Therefore, we propose the pharmacological modulation of P2X7R as a therapeutic approach against MDD symptoms.