Desarrollo y utilización de métodos computacionales en la mejora del proceso de obtención de nuevos fármacos

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-02-201

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Background and Aims We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL–apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Conclusions Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratificatio

Potency assessment of IFNγ-producing SARS-CoV-2- specific T cells from COVID-19 convalescent subjects

The development of new therapies for COVID-19 high-risk patients remains necessary to prevent additional deaths. Here, we studied the phenotypical and functional characteristics of IFN-γ producing-SARS-CoV-2-specific T cells (SC2-STs), obtained from 12 COVID-19 convalescent donors, to determine their potency as an off-the-shelf T cell therapy product. We found that these cells present mainly an effector memory phenotype, characterized by the basal expression of cytotoxicity and activation markers, including granzyme B, perforin, CD38, and PD-1. We demonstrated that SC2-STs could be expanded and isolated in vitro, and they exhibited peptide-specific cytolytic and proliferative responses after antigenic re-challenge. Collectively, these data demonstrate that SC2-STs can be a suitable candidate for the manufacture of a T cell therapy product aimed to treat severe COVID-19.This study was supported by the Department of University, Innovation and Digital Transformation of Government of Navarra, with the grant 0011-3597-2020-000006

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Background and Aims We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Conclusions Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.National Institutes of Health (R01DK123763, R01DK119437, HL151328, P30DK52574, P30DK56341, and UL1TR002345); Ministerio de Economía y Competitividad de España (SAF2017-88041-R); Ministerio de Economía y Competitividad de España for the Severo Ochoa Excellence Accreditation (SEV-2016-0644); CIBERehd (Biomedical Research Center in Hepatic and Digestive Diseases) and Netherlands Organization for Applied Scientific Research Program (PMC13 and PMC15); Spanish Carlos III Health Institute (PI15/01132 and PI18/01075); Miguel Servet Program (CON14/00129 and CPII19/00008); Fondo Europeo de Desarrollo Regional, CIBERehd, Department of Industry of the Basque Country (Elkartek: KK-2020/00008); La Caixa Scientific Foundation (HR17-00601); Liver Investigation: Testing Marker Utility in Steatohepatitis consortium funded by the Innovative Medicines Initiative Program of the European Union (777377), which receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA; Newcastle NIHR Biomedical Research Center; Czech Ministry of Health (RVO-VFN64165/2020); Fondo Nacional De Ciencia y Tecnología de Chile (1191145); and the Comisión Nacional de Investigación, Ciencia y Tecnología (AFB170005, CARE Chile UC); Agencia Nacional de Investigación y Desarrollo (ANID ACE 210009); European Union's Horizon 2020 Research and Innovation Program (825510)

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Receptor-Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio- and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking-based virtual screening technique. Some of the compounds identified have invitro activities in the low-to-medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.Fondo de Investigaciones Sanitarias (grant PI06/1250); Ministerio Ciencia e Innovación (grant CTQ-2010-20541-C03-03); Comunidad de Madrid (SBIO-0214-2006 BIPEDD and S2010 BMD-2457 BIPEDD2); Generalitat de Catalunya; Instituto de Salud Carlos III; AMAROUTOPeer Reviewe

Inhibidores de la enzima 06-alquilguanina-ADN-metil-transferasa para el tratamiento del cáncer

Fecha de solicitud: 10-12-2009.- Titular: Consejo Superior de Investigaciones Científicas (CSIC)The invention relates to the use of compounds derived from piperadinyl-methyl-tetrazole-quinolinone and derived from diphenyl-triazolo- pyrimidine as inhibitors of the reparative action of the DNA produced by enzyme O6-alkylguanine-DNA-methyltransferase, and to pharmaceutical compositions containing same, for the preparation of coadjuvant drugs for use in antitumour therapy using alkylating agents.La invención relaciona a la utilización de compuestos derivada de piperadinyl-metil-tetrazol-quinolinona y derivada de pirimidina del difenilo-triazolo como inhibidores de la acción reparativa de la DNA producida por la enzima O6-alkylguanine-DNA-methyltransferase, y a contener de las composiciones farmacéuticas mismo, para la preparación de los fármacos coadjuvant para el uso en terapia antitumour usando a agentes de alquilación.Peer reviewe

MM-ISMSA: An ultrafast and accurate scoring function for protein-protein docking

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r 2 ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations. © 2012 American Chemical Society.CICYT (SAF2009-13914-C02-02); Comunidad Autónoma de Madrid; AMAROUTO; Ministerio de Economía y Competividad; Ministerio de Educación y CienciaPeer Reviewe