Estudios computacionales de interacciones carbohidrato-proteína

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 16/06/2017Molecular modelling techniques have been extensively applied to the elucidation of glycan-protein interactions at the atomic level of several systems with biological and therapeutic interests, and have provided new insights for the understanding of the molecular recognition events underlying the biological functions of these systems. In this Thesis, the general objective is to elucidate the carbohydrate-protein interactions at the atomic level through computational techniques. In particular, the following systems have been studied: human galectins -1, -3, and -7, Pisum sativum lectin, Maackia amurensis seed lectin, and glycosyltransferase GalNAc-T2. The final aim is to provide new insights for the understanding of the molecular recognition events underlying the biological functions of these proteins...En esta Tesis Doctoral, se han empleado técnicas de modelado molecular para la elucidación a nivel atómico de las interacciones carbohidrato-proteína de varios sistemas con interés biológico y terapéutico. Estos estudios han permitido aportar nuevas perspectivas para la comprensión de los procesos de reconocimiento molecular implicados en las funciones biológicas de estos sistemas. En particular, se han estudiado los siguientes sistemas: galectinas humanas -1, -3 y -7, lectina de Pisum sativum, lectina de semillas de Maackia amurensis, y la glicosiltransferasa GalNAc-T2. El objetivo final es proporcionar nuevas perspectivas para la comprensión de los eventos de reconocimiento molecular que subyacen en las funciones biológicas de estas proteínas...Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEunpu

Precursors for cytochrome P450 profiling breath tests from an in silico screening approach

The family of cytochrome P450 enzymes (CYPs) is a major player in the metabolism of drugs and xenobiotics. Genetic polymorphisms and transcriptional regulation give a complex patient-individual CYP activity profile for each human being. Therefore, personalized medicine demands easy and non-invasive measurement of the CYP phenotype. Breath tests detect volatile organic compounds (VOCs) in the patients’ exhaled air after administration of a precursor molecule. CYP breath tests established for individual CYP isoforms are based on the detection of 13CO2 or 14CO2 originating from CYP-catalyzed oxidative degradation reactions of isotopically labeled precursors. We present an in silico work-flow aiming at the identification of novel precursor molecules, likely to result in VOCs other than CO2 upon oxidative degradation as we aim at label-free precursor molecules. The ligand-based work-flow comprises five parts: (1) CYP profiling was encoded as a decision tree based on 2D molecular descriptors derived from established models in the literature and validated against publicly available data extracted from the DrugBank. (2) Likely sites of metabolism were identified by reactivity and accessibility estimation for abstractable hydrogen radical. (3) Oxidative degradation reactions (O- and N-dealkylations) were found to be most promising in the release of VOCs. Thus, the CYP-catalyzed oxidative degradation reaction was encoded as SMIRKS (a programming language style to implement reactions based on the SMARTS description) to enumerate possible reaction products. (4) A quantitative structure property relation (QSPR) model aiming to predict the Henry constant H was derived from data for 488 organic compounds and identifies potentially VOCs amongst CYP reaction products. (5) A blacklist of naturally occurring breath components was implemented to identify marker molecules allowing straightforward detection within the exhaled air.peer-reviewe

Minimizing the entropy penalty for ligand binding: lessons from the molecular recognition of the histo blood-group antigens by human galectin-3

6 p.-5 fig.-2 tab.Ligand conformational entropy plays an important role in carbohydrate recognition events. Glycans are characterized by intrinsic flexibility around the glycosidic linkages, thus in most cases, loss of conformational entropy of the sugar upon complex formation strongly affects the entropy of the binding process. By employing a multidisciplinary approach combining structural, conformational, binding energy, and kinetic information, we investigated the role of conformational entropy in the recognition of the histo blood‐group antigens A and B by human galectin‐3, a lectin of biomedical interest. We show that these rigid natural antigens are pre‐organized ligands for hGal‐3, and that restriction of the conformational flexibility by the branched fucose (Fuc) residue modulates the thermodynamics and kinetics of the binding process. These results highlight the importance of glycan flexibility and provide inspiration for the design of high‐affinity ligands as antagonists for lectins.We thank Agencia Estatal de Investigacion and ISCIII of Spain and the European Research Council for financial support.Peer reviewe

Abacavir increases purinergic P2X7 receptor activation by ATP: does a pro-inflammatory synergism underlie its cardiovascular toxicity?

16 p.-9 fig.-1 tab.The cardiovascular toxicity of Abacavir is related to its purinergic structure. Purinergic P2X7-receptors (P2X7R), characterized by activation by high concentrations of ATP and with high plasticity, seem implicated. We appraise the nature of the interplay between Abacavir and P2X7R in generating vascular inflammation. The effects of Abacavir on leukocyte-endothelium interactions were compared with those of its metabolite carbovir triphosphate (CBV-TP) or ATP in the presence of apyrase (ATP-ase) or A804598 (P2X7R-antagonist). CBV-TP and ATP levels were evaluated by HPLC, while binding of Abacavir, CBV-TP and ATP to P2X7R was assessed by radioligand and docking studies. Hypersensitivity studies explored a potential allosteric action of Abacavir. Clinical concentrations of Abacavir (20 µmol/L) induced leukocyte-endothelial cell interactions by specifically activating P2X7R, but the drug did not show affinity for the P2X7R ATP-binding site (site 1). CBV-TP levels were undetectable in Abacavir-treated cells, while those of ATP were unaltered. The effects of Abacavir were Apyrase-dependent, implying dependence on endogenous ATP. Exogenous ATP induced a profile of proinflammatory actions similar to Abacavir, but was not entirely P2X7R-dependent. Docking calculations suggested ATP-binding to sites 1 and 2, and Abacavir-binding only to allosteric site 2. A combination of concentrations of Abacavir (1 µmol/L) and ATP (0.1 µmol/L) that had no effect when administered separately induced leukocyte-endothelium interactions mediated by P2X7R and involving Connexin43 channels. Therefore, Abacavir acts as a positive allosteric modulator of P2X7R, turning low concentrations of endogenous ATP themselves incapable of stimulating P2X7R into a functional proinflammatory agonist of the receptor.This work was supported by Ministerio de Economía y Competitividad and the European Regional Development fund of the European Union (FEDER) (SAF2015–67678-R, RTI2018-094436-B-I00 and CTQ2017-88353-R), Ministerio de Sanidad y Consumo (CB06/04/0071, CIBERehd) and Generalitat Valenciana (PROMETEOII/2014/035 and PROMETEO 2018/141), along with an unrestricted grant from GILEAD S.L. VCD and ASL were funded by VALI + D program from Generalitat Valenciana (grants number ACIF/2015/316 and ACIF/2016/119, respectively) and PGM by FPU program from Ministerio de Educación, Cultura y Deporte (grant number FPU16/06064) and MABR by FPU program from Ministerio de Ciencia, Innovación y Universidades (grant number FPU17/04249).Peer reviewe

Computational approaches to toll-like receptor 4 modulation

24 p.-10 fig.Toll-like receptor 4 (TLR4), along with its accessory protein myeloid differentiation factor 2 (MD-2), builds a heterodimeric complex that specifically recognizes lipopolysaccharides (LPS),which are present on the cell wall of Gram-negative bacteria, activating the innate immune response.Some TLR4 modulators are undergoing preclinical and clinical evaluation for the treatment of sepsis,inflammatory diseases, cancer and rheumatoid arthritis. Since the relatively recent elucidation of the X-ray crystallographic structure of the extracellular domain of TLR4, research around this fascinating receptor has risen to a new level, and thus, new perspectives have been opened. In particular,diverse computational techniques have been applied to decipher some of the basis at the atomic level regarding the mechanism of functioning and the ligand recognition processes involving the TLR4/MD-2 system at the atomic level. This review summarizes the reported molecular modeling and computational studies that have recently provided insights into the mechanism regulating the activation/inactivation of the TLR4/MD-2 system receptor and the key interactions modulating the molecular recognition process by agonist and antagonist ligands. These studies have contributed to the design and the discovery of novel small molecules with promising activity as TLR4 modulators.This work was supported by the Spanish Ministry for Economy and Competitiveness (MINECO) Grant CTQ2014-57141-R and PhD fellowship BES-2015-071588 and by the European Commission granted GLYCOPHARM Marie Curie ITN PITN-GA-2012-317297 (www.glycopharm.eu) and TOLLerant H2020-MSC-ETN-642157 (www.tollerant.eu) projects.Peer reviewe

New Therapeutic Strategies for Osteoarthritis by Targeting Sialic Acid Receptors

© 2020 by the authors.Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing α-2,6-linked sialic acids to those that contain α-2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of α-2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the α-2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting α-2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.This work was funded by the Spanish Society for Rheumatology (SER-2013) and the Spanish Society for Research on Bone and Mineral Metabolism (FEIOMM-2016) (to M.D.M.) and by the grant PI13/00591, PI16/00035, and PI19/00145 from the Health Institute “Carlos III” (ISCIII, Spain) and was cofinanced by the European Regional Development Funds, “A way of making Europe” from the European Union (to M.D.M.). Paula Carpintero-Fernández has a postdoctoral fellowship from Xunta de Galicia (IN606B 2017/014) Two predoctoral fellowships from Xunta de Galicia to Marta Varela-Eirin (ED481A 2015/188) and Raquel Gago-Fuentes (PRE/2012/165) and from University of A Coruña—INDITEX and CICA-INIBIC (AGRUP2015/05). The Spanish Ministry for Economy and Competitiveness (MINECO) grants CTQ2014-57141-R and CTQ2017-88353-R (to S.M.-S.) is gratefully acknowledged.Peer reviewe

Characterisation of the Dynamic Interactions between Complex N-Glycans and Human CD22

CD22 (Siglec-2) is a B-cell surface inhibitory protein able to selectively recognize sialylated glycans, dampening autoimmune responses against self-antigens. We here characterize the dynamic recognition of complex-type N-glycans by human CD22, by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide novel molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid-galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results will enable the development of additional therapeutics able to modulate the activity of h-CD22 in autoimmune diseases and B-cell derived malignancies