20 research outputs found

    Study of the interaction between sialic acid-binding immunoglobulin-type lectins (Siglec) and sialylated glycans for the development of a new generation of immunomodulators.

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    Glycans and complementary glycan-binding proteins represent essential components in the control of both innate and adaptive immunity. Sialic acids are a family of sugars found on the terminal end of mammalian glycoconjugates; they able to act as marker of self in the immune system, as such residues are absent in most microbes. Sialic acid-binding immunoglobulin-like lectins, or Siglecs, are cell surface receptors that recognize sialic acids and are known to modulate immune responses, influencing almost every cell in the hematopoietic system. Siglecs are involved in events like cell adhesion and signaling, inhibition or regulation of the immune cell activation, all mediated by the interaction with sialylated ligands. Sialic acid-Siglec interactions have been associated with a broad spectrum of diseases, stretching from autoimmunity to neurodegeneration and cancer. Thus, strategies for a rational modulation of the interactions between Siglecs and sialylated glycans in pathophysiological processes exhibit a great therapeutic potential. In this context, the present thesis project aimed at the study of the interaction between Siglecs and their cognate sialic acid containing ligands, to disclose the key recognition events underlining host immune suppression or activation. To this end, a multidisciplinary approach combining advanced technologies as ligand-based NMR techniques, including STD-NMR and tr-NOESY, biophysical binding assays and computational methodologies, such as homology modelling docking and MD simulations, was applied to provide an atomistic depiction of the interaction interfaces between various sialoglycans and their receptors. The described strategy has been employed to characterize the binding features of several receptors of the Siglecs family, namely CD22/Siglec-2, Siglec-10 and Siglec-7. CD22 is a B-cell surface inhibitory protein capable of selectively -(2,6) linked sialylated glycans, thus dampening autoimmune responses against self-antigens. The characterization of complex-type N-glycans by CD22 allowed to describe the conformational behavior of the flexible ligands; the formation of CD22 homo-oligomers on the B-cell surface was also addressed. Furthermore, it was provided a global vision of how the most diffuse neuraminic acid forms of sialylated N-glycans are accomodated in the binding pocket of CD22. Moreover, the elucidation of the binding epitope of a synthetic sialo-mimetic upon CD22 interaction afforded new hints for the design and synthesis of high-affinity ligands of therapeutic relevance against B-cell derived malignancies. Then, the Siglec-10, an inhibitory receptor that recognize 2,3 and -linked sialoglycans was studied, thus providing the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. After that, Siglec-7, a well-established inhibitory receptor that is primarily located on natural killer where it acts as inhibitor of cancer cells cytotoxicity via sialylated ligands binding, has been characterized in the interplay with the oncogenic pathogen F. nucleatum. Indeed, the presence of sialylated lipopolysaccharide (LPS) on certain F. nucleatum strains, hinted that it may have a significant role at the immune interface. The interaction between Siglec-7 and the O-polysaccharide chain from the LPS of F. nucleatum 10953 strain has been depicted, thus delineating a structural binding model that might contribute to explain the etiological role of F. nucleatum in carcinogenesis. A similar approach was employed to other sialoglycan- related systems, i. e. to dissect the mechanism of sialic acid recognition and hydrolysis by mumps virus hemagglutinin neuraminidase, a viral glycoprotein that plays key roles in virus entry and infection; and to assess the binding of the human macrophage galactose-type lectin (MGL) in the interplay with lipooligosaccharide of E. coli strain R1. In conclusion, the structural and functional characterization of Siglec- sialylated glycans interaction have allowed the analysis, at a molecular level, of crucial feature of 3D complexes, highlighting the molecular determinants involved in recognition and binding events, that will aid for the development or optimization of molecules for therapeutic targeting of the Siglecs

    Novel saturation transfer difference (STD) NMR approaches to understand biologically relevant protein-carbohydrate interactions

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    Saturation transfer difference (STD) NMR spectroscopy is a powerful NMR technique extensively used to obtain epitope maps of ligands binding to biologically relevant protein receptors. This allows to reveal semi-quantitative structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. In this thesis, the main effort has been put to develop two novel implementations of STD NMR, aimed at elucidating the surroundings of the ligand (i.e., the amino acids lining the binding pocket, or an adjacent bound ligand) in biologically relevant complexes. First, we report the development of the novel “DiffErential EPitope mapping STD NMR” (DEEP‐STD NMR), a method producing differential epitope maps through i) differential frequency and/or ii) differential solvent (D2O/H2O) STD NMR experiments. These two approaches provide complementary information on the architecture of the binding pocket. The second novel method we propose is “Inter-ligand STD NMR” (IL-STD NMR), which relies on on-ligand differential frequency STD NMR to detect contacts between ligands bound to adjacent sites of a receptor. These novel STD NMR methodologies, in combination with traditional STD NMR and computational tools, have been applied to the study of two systems: the interactions of Cholera Toxin subunit B (CTB) with a set of promising inhibitors; and the interactions of an intramolecular trans-sialidase from Ruminococcus gnavus, a gut microbiota symbiont, with a set of mucin-related sialylated ligands. In the first study, we discovered the existence of a hitherto unknown binding subsite in the GM1 binding site of CTB. In the second study, we provided the first 3D molecular model of a Michaelis complex for an IT-sialidase. In both cases, we demonstrate that our newly developed approaches increase the level of resolution of STD NMR, widening its potential to impact the field of ligand design for biologically relevant receptors

    Resonancia magnética nuclear aplicada al estudio sobre el reconocimiento molecular de ligando con fucosa mediado por el receptor DC-SIGN

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    La presente tesis doctoral se dirige al estudio de la proteína humana DC-SIGN desde una perspectiva estructural, atendiendo a aquellos aspectos que, a nivel molecular, determinan su interacción con sus ligandos de tipo carbohidrato (conformación, interacciones intermoleculares (vdW)…). En concreto, el trabajo aquí expuesto se ha enfocado en el análisis de los procesos de reconocimiento en los que participan ligandos que contienen el monosacárido natural L-fucosa en su estructura. El interés por estudiar dicha proteína se ha acrecentado desde comienzos de este siglo, cuando DC-SIGN fue definitivamente identificada como un receptor de membrana de la familia de las C-type lectins y, más importante, se demostró su implicación directa en los procesos de infección por VIH. A partir de aquí, sucesivos trabajos permitieron subrayar la importancia de este receptor en otros procesos infecciosos que incluían patógenos de todo tipo, ya fueran virus (Ébola), bacterias (M. tuberculosis), hongos (C. albicans) o parásitos (S. mansoni). Desde un punto de vista estructural, es bien sabido que DC-SIGN reconoce e interacciona con oligosacáridos presentes en la superficie externa de estos agentes patógenos. El abanico de oligosacáridos que esta proteína es capaz de reconocer es bastante amplio, aunque es bien sabido que todos ellos contienen fundamentalmente los monosacáridos D-manosa y L-fucosa, con los que interacciona directamente. La interacción con D-manosa ha sido ampliamente estudiada a nivel molecular, especialmente mediante cristalografía de rayos X, lo cual ha permitido disponer de varios modelos que describen con un alto grado de detalle cómo ocurre el reconocimiento de este azúcar y cómo influyen tanto la estructura de la proteína como la del propio oligosacárido en dicho reconocimiento. En cambio, aunque la interacción de DC-SIGN con L-fucosa ha sido estudiada en paralelo, mucha de la información existente referente al reconocimiento de oligosacáridos fucosilados no está completamente justificada desde un punto de vista estructural, lo que ha dificultado en muchos casos la búsqueda de patrones estructurales que permitan entender bien cómo tiene lugar el reconocimiento..

    Desarrollo y validación experimental de un método para la obtención de estructuras de complejos ligando/receptor basado en Resonancia Magnética Nuclear

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    Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 14-03-201

    Metodologías innovadoras basadas en 19F-RMN de ligando y técnicas computacionales para el estudio de procesos de reconocimiento molecular azúcar-lectina

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 08-06-2021Carbohydrates play a central role in a large myriad of biological processes. They are found in all living organisms in nature, participating in different functions ranging from their use as energy source or as structural fragments, to infection-related processes in complex organisms. In vertebrates, they are located both in the cell surface and in the extracellular space, forming very diverse and intricate structures, but they are also present in the nucleus and cytoplasm of eukaryotic cells bound to proteins (glycoproteins). Their location almost ubiquitous in the organism confers them the capacity of mediate in a large number of ‘communication’ processes with other entities, for instance, in cell-cell, cell-molecule and cell-matrix interactions. In addition, carbohydrates intervene in molecular recognition processes between different organisms, such as the pathogen and parasite recognition by host cells...Los carbohidratos juegan un papel fundamental en una enorme variedad de procesos biológicos. Se encuentran en todos los organismos vivos en la naturaleza, donde intervienen en funciones que abarcan desde su uso como fuente de energía o como fragmentos estructurales, hasta procesos de infección en organismos superiores. En vertebrados, se localizan tanto en la superficie celular como en el espacio extracelular, formando estructuras muy diversas y complejas, pero también están presentes en el núcleo y citoplasma de células eucariotas unidos a proteínas (glicoproteínas). Su localización casi universal en el organismo les confiere la capacidad de intervenir en un gran número de procesos de ‘comunicación’ con otras entidades, por ejemplo, interacciones intercelulares, célula-molécula y célula-matriz extracelular. Además, los carbohidratos median procesos de reconocimiento molecular entre distintos organismos, como el reconocimiento de patógenos y parásitos por la célula de un huésped...Fac. de FarmaciaTRUEunpu

    Structural glycobiology by NMR and molecular modelling: ligand recognition by the carbohydrate active proteins LYVE-1, SseK1/2 and PsLBP

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    The term carbohydrate-active protein (CAP) encompasses the group of proteins that either act as carbohydrate receptors (e.g. lectins) or enzymatically catalyse reactions involving carbohydrates as at least one of their substrates (e.g. glycosyltransferases). The fundamental importance of CAPs has only been realised over the past few decades, with carbohydrates playing a profound role in cellular signalling, adhesion and migration. Furthermore, carbohydrates are routinely used by pathogens in immune evasion and to modify host function, and changes in glycosylation patterns are associated with a range of disease states, including cancer and inflammation. However, the study of CAP-carbohydrate interactions has been challenging, in part due to the inherently low affinity of many CAP-carbohydrate interactions that precluded detection by many techniques and make it difficult to obtained experimentally derived structures of their complexes. Fortunately, STD NMR spectroscopy is ideally suited to detecting weak interactions of this nature and provides structural information about the interaction through ligand epitope mapping. Furthermore, quantitative analysis of STD intensities allows three-dimensional models of the validated in the solution state, whether these models be derived from experiment or molecular modelling. Within this thesis, a combination of STD NMR spectroscopy and molecular modelling has been used to unravel structural and dynamic detail of CAP-ligand interactions in three biologically or industrially relevant systems - (1) CD44/LYVE-1 which may play a role in cell trafficking across the lymphatics in cancer; (2) PsLBP which may lead to new developments in the field of enzymatic carbohydrate synthesis; (3) SseK1/2 which exhibit a novel enzymatic mechanism involving glycosylation of arginine residues and may be involved in Salmonella virulence

    QCSPScore: a new scoring function for driving protein-ligand docking with quantitative chemical shifts perturbations

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    Through the use of information about the biological target structure, the optimization of potential drugs can be improved. In this work I have developed a procedure that uses the quantitative change in the chemical perturbations (CSP) in the protein from NMR experiments for driving protein-ligand docking. The approach is based on a hybrid scoring function (QCSPScore) which combines traditional DrugScore potentials, which describe the interaction between protein and ligand, with Kendall’s rank correlation coefficient, which evaluates docking poses in terms of their agreement with experimental CSP. Prediction of the CSP for a specific ligand pose is done efficiently with an empirical model, taking into account only ring current effects. QCSPScore has been implemented in the AutoDock software package. Compared to previous methods, this approach shows that the use of rank correlation coefficient is robust to outliers. In addition, the prediction of native-like complex geometries improved because the CSP are already being used during the docking process, and not only in a post-filtering setting for generated docking poses. Since the experimental information is guaranteed to be quantitatively used, CSP effectively contribute to align the ligand in the binding pocket. The first step in the development of QCSPScore was the analysis of 70 protein-ligand complexes for which reference CSP were computed. The success rate in the docking increased from 71% without involvement of CSP to 100% if CSP were considered at the highest weighting scheme. In a second step QCSPScore was used in re-docking three test cases, for which reference experimental CSP data was available. Without CSP, i.e. in the use of conventional DrugScore potentials, none of the three test cases could be successfully re-docked. The integration of CSP with the same weighting factor as described above resulted in all three cases successfully re-docked. For two of the three complexes, native-like solutions were only produced if CSP were considered.Conformational changes in the binding pockets of up to 2 Å RMSD did not affect the success of the docking. QCSPScore will be particularly interesting in difficult protein-ligand complexes. They are in particular those cases in which the shape of the binding pocket does not provide sufficient steric restraints such as in flat protein-protein interfaces and in the virtual screening of small chemical fragments.Durch die Verwendung von Information über die biologische Zielstruktur kann die Optimierung potentieller Wirkstoffe verbessert werden. Im Rahmen dieser Arbeit habe ich ein Verfahren entwickelt, das quantitativ die Veränderung der Chemischen Verschieben (CSP) im Protein aus NMR-Experimenten für das Protein-Ligand-Docking verwendet. Der Ansatz basiert auf einer Hybridbewertungsfunktion (QCSPScore) und kombiniert herkömmliche DrugScore-Potentiale, welche die Wechselwirkung zwischen Protein und Ligand beschreiben, mit dem Rangkorrelationskoeffizienten nach Kendall, der die Dockingposen hinsichtlich ihrer Übereinstimmung mit experimentellen CSP. Die Vorhersage der CSP für einen bestimmten Liganden geschieht effizient mit einem empirischen Modell, wobei nur Ringstromeffekte berücksichtigt werden. QCSPScore wurde in das AutoDock Softwarepaket implementiert. Im Vergleich zu früheren Verfahren zeigt dieser Ansatz, dass die Verwendung des Rangkorrelationskoeffizienten robuster ist gegenüber Ausreißern in den vorhergesagten CSP. Außerdem ist die Vorhersage nativ-ähnlicher Komplexgeometrien verbessert, da die CSP bereits während des Docking-Prozesses eingesetzt werden, und nicht erst in einem nachträglichen Filter für generierte Dockingposen. Da die experimentelle Informationen quantitativ benutzt werden wird sichergestellt, dass die CSP effektiv dazu beitragen, den Liganden in der Bindetasche auszurichten. Der erste Schritt bei der Entwicklung des QCSPScore war die Analyse von 70 Protein-Ligand-Komplexen, für die als Referenz CSP vorhergesagt wurden. Die Erfolgsrate im Docking erhöhte sich von 71 %, ohne Einbeziehung von CSP, auf 100 %, wenn CSP mit höchster Gewichtung mit einbezogen wurden. Die globale Optimierung auf der kombinierten Docking-Energiehyperfläche ist also erfolgreich. In einem zweiten Schritt wurde QCSPScore zum Docking dreier Testfälle verwendet, für die als Referenz experimentelle CSP zur Verfügung standen. Ohne CSP, d.h. bei der Verwendung von herkömmlichen DrugScore-Potentialen, konnte keiner der drei Testfälle erfolgreich gedockt werden. Die Einbeziehung von CSP mit dem selben hohen Gewichtungsfaktor wie oben führte in allen drei Fällen zu erfolgreichen Docking-Ergebnissen. Für zwei der drei Komplexe wurden zudem nur bei Einbeziehung der experimentellen Information nativ-ähnliche Geometrien vorhergesagt. Konformationelle Änderungen der Bindetasche bis zu 2 Å RMSD beeinträchtigen den Erfolg des Dockings nicht. Ich bin davon überzeugt, dass mein Verfahren besonders für Protein-Ligand-Komplexe interessant sein wird, für die die Vorhersage nativ-ähnlicher Komplexe bislang schwierig war. Das sind insbesondere solche Fälle, in denen die Form der Bindetasche zur Vorhersage des Komplexes nicht ausreichend, wie das bei flachen Protein-Protein-Wechselwirkungsregionen oder beim virtuellen Screening kleiner Fragmente der Fall ist

    Development and application of NMR methods for challenges in drug discovery

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    Nuclear Magnetic Resonance Spectroscopy

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    Nuclear Magnetic Resonance (NMR) spectroscopy is a nondestructive technique that can be used to characterize a wide variety of systems. Sustained development of both methodology and instrumentation have allowed NMR to evolve as a powerful technology, with applications in pure sciences, medicine, drug development, and important branches of industry. NMR provides precise structural information down to each atom and bond in a molecule, and is the only method for the determination of structures of molecules in a solution. This book compiles a series of articles describing the application of NMR in a variety of interesting scientific challenges. The articles illustrate the versatility and flexibility of NMR
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