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Scaffold optimisation and activity evaluation of tetra-pseudoglycosylated MBL antagonists
No full textMannose Binding Lectin (MBL) is a circulating multimeric calcium dependent (C-lectin) protein that acts as a first-line host defence by selectively recognizing and binding to polyglycosylated surfaces of invading pathogens or damaged self cells.1MBL is also able to modulate inflammation and it is likely involved in reperfusion damage in acute stroke,2 therefore representing an attractive target for the development of new drugs against this disease. Our groups have already reported that pseudoglycosylated tetravalent dendrimers based on polyester scaffolds are good antagonists of MBL, with kd values in the low micromolar range. They were shown to supply a protective effect in a mouse model of brain stroke, reducing the reperfusion damage with a surprisingly wide therapeutic window.3
Unfortunately, these constructs proved to be rather chemically unstable: the polyester scaffold does not survive silica gel chromatography in either direct or reverse phase and slowly hydrolyses in water solution at physiological pH (27% hydrolysis in 6h). Here we present the synthesis of new dendrimers characterised by a stabilised version of the scaffold that allows to obtain still water soluble compounds with a grater chemical stability. Results of in vitro SPR binding assays of the new dendrimers to MBL will also be discussed
GLYCOMIMETIC ANTAGONISTS OF C-TYPE LECTINS: IMPROVING LIGAND POTENCY AND MULTIVALENT PRESENTATION
Get PDFLe lectine di tipo C sono recettori calcio dipendenti in grado di riconoscere in modo selettivo e di legarsi a carboidrati esposti da patogeni e cellule danneggiate, agendo quindi come un’importante meccanismo di prima difesa. Tuttavia, lectine di tipo C possono essere responsabili della patogenesi di infezioni e malattie infiammatorie, costituendo così un promettente bersaglio terapeutico.
Le interazioni proteina-carboidrato sono generalmente deboli, con costanti di dissociazione nell’ordine del millimolare. Tale problema di bassa ffinità è stato risolto in Natura ricorrendo alla multivalenza: lectine di tipo C sono solitamente recettori oligomerici che interagiscono con superfici poliglicosilate, di conseguenza concomitanti interazioni multiple determinano un legame ad alta affinità.
Questo lavoro di tesi è stato dedicato alla sintesi e identificazione di forti antagonisti di lectine di tipo C. In particolare, la strategia adottata si è basta sulla combinazione di composti glicomimetici come ligandi attivi e resistenti all’azione di enzimi e sulla loro presentazione multivalente.
Una prima parte del seguente lavoro è stato incentrato sulla stabilizzazione di antagonisti precedentemente sviluppati contro la Mannose Binding Lectin (MBL) , una lectina di tipo C implicata nel danno da riperfusione in seguito ad ictus emorragico. Glicodendroni tetravalenti sono stati sintetizzati tramite un approccio efficiente e modulare. La strategia adottata ha permesso la funzionalizzazione a livello del punto focale del dendrone consentendo un rapido accesso a composti a valenza superiore, come dimostrato dalla sintesi di un glicodendrimero 16-valente. È stato possibile effettuare una caratterizzazione di tutti i dendroni e dendrimeri, che si sono rivelati solubili in acqua ed estremamente stabili sia in soluzione acquosa, sia durante purificazione tramite cromatografia (sia fase diretta che fase inversa). L’affinità dei composti è stata verificata nei confronti dell’isoforma murina MBL-C tramite un saggio SPR competitivo, che ha confermato una alta capacità di legare la proteina fortemente dipendente dalla valenza dei ligandi.
Un’ulteriore parte del lavoro è stato volto alla sintesi di inibitori della Dendritic Cell-Specific ICAM-Grabbing Non-Integrin (DC-SIGN), una lectina di tipo C sfruttata da vari agenti patogeni per dare inizio ad infezione. Nuovi antagonisti di DC-SIGN sono stati sviluppati tramite una progettazione razionale. Vista l’alta affinità raggiunta da agenti chelanti divalenti ed esavalenti, è stato progettato e sintetizzato un glicodendrimero dodecavalente a forma di croce capace di legare simultaneamente i quattro siti di riconoscimento di DC-SIGN. Studi per valutarne l’affinità nei confronti di DC-SIGN sono attualmente in corso.
Infine, in collaborazione col gruppo del Dr. Niels C. Reichardt presso CIC biomaGUNE (San Sebastian, Spain) è stato intrapreso lo sviluppo di una nuova metodologia volta all’identificazione di nuovi ligandi glicomimetici per lectine di tipo C. Una piccola libreria di composti glicomimetici è stata sintetizzata sfruttando la tecnologia dei microarray di glicani. In particolare, l’utilizzo di microarray analizzabili via MALDI-TOF MS ha permesso la sintesi on-chip di glicani non naturali tramite glicosilazione enzimatica con donatori funzionalizzabili tramite Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). Al fine di generare una libreria di mimetici del trisaccaride LewisX tramite tale metodologia, è stata eseguita la sintesi di un Fucosio-GDP derivato contenente un gruppo azidico. Come da noi dimostrato, questo composto viene accettato come donatore di legame glicosidico in modo eccellente dall’enzima α-1,3-fucosiltransferasi da H. pylori e la sintesi di mimetici di LewisX è tuttora in corso presso il centro di ricerca CIC biomaGUNE.C-Type lectin receptors are calcium dependent proteins able to selectively recognize and bind to carbohydrates exposed by invading pathogens and damaged cells, thus acting as important first-line host defence. However, C-type lectins activity can be also responsible for the pathogenesis of infections and inflammatory diseases, thus representing a class of promising therapeutic targets.
Protein-carbohydrate interactions are generally weak interactions, with dissociation constants lying in the millimolar range. This problem has been solved by Nature exploiting multivalency: C-type lectins are usually oligomeric receptors interacting with polyglycosylated surfaces, thus concomitant multiple interactions lead to high affinity binding.
This thesis work has been devoted to the synthesis and identification of potent C-type lectin antagonists. In particular, our strategy was based on the combination of glycomimetic compounds as active and enzymatically stable ligands and on their multivalent presentation.
The first part of the thesis work was focused on the synthesis of stabilised versions of previously developed antagonists for the Mannose Binding Lectin (MBL), a C-type lectin involved in the reperfusion damage after acute ischemic stroke. Tetravalent glycodendrons were synthesized following an efficient modular approach, which allowed functionalization at the dendron focal point and ready access to higher valency compounds, as demonstrated with the synthesis of a 16-valent glycodendrimer. All the compounds were fully characterized and showed complete water solubility and remarkable stability in water solution and under chromatographic purification (both direct and reverse phase). The binding affinity of the glycodendons and glycodendrimer was assessed towards the murine isoform MBL-C by an SPR competition assay, confirming high binding affinity and valency-dependent avidity.
A second part of the work was addressed to the synthesis of inhibitors for the Dendritic Cell-Specific ICAM-Grabbing Non-Integrin (DC-SIGN), a C-type lectin exploited by several pathogens to initiate infections. In the development of new DC-SIGN antagonists, a rational design approach was adopted. Driven by the high affinity reached by divalent and hexavalent DC-SIGN chelating agents, a cross-shaped 12-valent glycodendrimer able to perform as a tetradentate ligand and simultaneously bind to the four DC-SIGN binding sites was designed and successfully synthesized and characterized. The affinity towards DC-SIGN is still under study.
Finally, the development of a methodology aimed to identify potent C-type lectin glycomimetic ligands was undertaken in collaboration with the group of Dr. Niels C. Reichardt at CIC biomaGUNE (San Sebastian, Spain). A small library of glycomimetics was synthesized exploiting glycan microarray technology. In particular, the use of recently described MALDI-TOF MS detectable microarrays allowed us the on-chip synthesis of unnatural glycan through enzymatic glycosylation with clickable modified sugar donors followed by Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). In the attempt of applying this strategy to the generation of LewisX trisaccharide mimics, the synthesis of a clickable Fucose-GDP sugar donor was accomplished. This derivative proved to be readily accepted by α-1,3-fucosyltransferase from H. pylori and the synthesis of LewisX mimics is currently on going at CIC biomaGUNE
Scaffold optimization of tetravalent C-lectin antagonists
No full textIn recent years our group, in collaboration with the Rojo group (Glycosystems Laboratory, IIQ-CSIC, Sevilla), has demonstrated that tetravalent polyester dendrimers[1] functionalised with glycomimetic pseudo-dimannoside and pseudo-trimannoside ligands work as good inhibitors of the C-type lectins DC-SIGN[1] and MBL[2].
MBL is a circulating C-type lectin that has crucial role in first line of host defense. MBL recognizes polyglycosylated cell surfaces present both on pathogens and self damaged cells and has great importance in the activation of complement, promotion of opsonophagocytosis, promotion of apoptosis and also modulation of inflammation.[3] Recent studies have shown that the capability of MBL in generating an inflammatory environment may be a main cause of the reperfusion damage that occurs after a stroke event, highlighting MBL as a promising target for this pathology.[2,4]
The antagonists that we developed so far are able to bind MBL with dissociation constant in the low micromolar range (SPR binding assays). Moreover, the inhibitor 1 bearing as a ligand the pseudo-trimannoside 2 (Figure 1) was tested in vivo and showed a great capability of reducing the ischemic volume in mice with an uncommon wide therapeutic window.[2]
The main drawback of this construct consists in its limited half-life in plasma, due to the instability of the succinyl ester which also determines the chromatographic lability of the dendron. Here we present an attempt in the optimization of this scaffold. We are particularly interested in obtaining a product that is still soluble in water and no longer sensitive to hydrolysis and therefore characterised by easier purification procedures and chemical and enzymatic stability.
Our results will be shown in the poster.
References:
1. J. Luczkowiak, S. Sattin, I. Sutkecičiūtė, J. J. Reina, M. Sánchez-Navarro, M. Thépaut, L. Martínez-Prats, A. Daghetti, F. Fieschi, R. Delgado, A. Bernardi, J. Rojo Bioconjugate Chem. 2011, 22, 1354.
2. F. Orsini, P. Villa, S. Parrella, R. Zangari, E. R. Zanier, R. Gesuete, M. Stravalaci, S. Fumagalli, R. Ottria, J. J. Reina, A. Paladini, E. Micotti, R. Ribeiro-Viana, J. Rojo, V. I. Pavlov, G. L. Stahl, A. Bernardi, M. Gobbi, M.-G. De Simoni Circulation 2012, 126, 1484.
3. M. W. Turner Molecular Immunology 2003, 40, 423; b) A. Cervera, A. M. Planas, C. Justicia, X.Urra, J. C. Jensenius, F. Torres, F. Lozano, A. Chamorro PLOS ONE 2010, 5, e8433.
4. R. Gesuete, C. Storini, A. Fantin, M. Stravalaci, E. R. Zanier, F. Orsini, H. Vietsch, M. L. M. Mannesse, B. Ziere, M. Gobbi, M.-G. De Simoni Annals of Neurology 2009, 66, 332
Scaffold optimization of a tetravalent C-lectin antagonist
No full textC-type lectins constitute a class of proteins able to selectively recognize carbohydrates and bind to polyglycosilated surfaces, which are considered as an interesting pharmaceutical target for their involvement in many physiological and pathological processes.1
In the past few years, in collaboration with the Rojo group (Glycosystems Laboratory, IIQ-CSIC, Sevilla), we demonstrated that two polyester tetravalent dendrons 1a,b functionalized either with pseudo-biomannoside 2 or pseudo-trimannoside 3 ligands (Fig. 1) are good antagonists of the C-lectins DC-SIGN2 and MBL3, which are respectively involved in HIV infection and ischemic reperfusion damage.
Despite the promising results obtained both in in vitro and in vivo experiments, the two dendrons showed to suffer from high chemical instability. Here we present the synthesis of two analogous tetraamide dendrons characterized by an optimized scaffold that led to much more stable and still water soluble constructs. Preliminary results of biological assays will also be shown
Scaffold optimisation and activity evaluation of multimeric MBL antagonists
No full textMannose Binding Lectin (MBL) is a circulating multimeric calcium dependent (C-lectin) protein that acts as a first-line host defence by selectively recognizing and binding to polyglycosylated surfaces of invading pathogens or damaged self cells.1 MBL is also able to modulate inflammation and it is likely involved in reperfusion damage in acute stroke,2 therefore representing an attractive target for the development of new drugs against this disease.
Our groups have already reported that pseudoglycosylated tetravalent dendrons based on a polyester scaffold such as 1a,b are good antagonists of MBL, with kd values in the low micromolar range. They were shown to supply a protective effect in a mouse model of brain stroke, reducing the reperfusion damage with a surprisingly wide therapeutic window.3 Unfortunately, these constructs proved to be rather chemically unstable: the polyester scaffold does not survive silica gel chromatography in either direct or reverse phase and slowly hydrolyses in water solution at physiological pH (27% hydrolysis in 6h).
Here we present the synthesis of two new dendrons characterised by an optimised scaffold that allows to obtain still water soluble compounds with an improved chemical stability. Preliminary results of in vitro SPR binding assays of the new dendrons to MBL will also be discussed
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
On-chip synthesis of MALDI-TOF MS detectable glycan microarrays
No full textGlycan microarrays are a versatile tool for the identification of endogenous ligands and effective inhibitors for lectins, proteins that selectively bind to carbohydrates. These devices are usually obtained through the covalent immobilization on a surface of a library of glycans which are screened towards a target lectin, revealing the entity of the interaction by fluorescence spectroscopy or by SPR technique.1
In recent years, Reichardt and co-workers have demonstrated that glycans microarrays of compounds absorbed through hydrophobic interactions on a lipid-functionalized ITO support enable analysis of the array by MALDI-TOF MS spectrometry (Figure).2 Here we show that this technology also allows on-chip synthesis of unnatural glycan through enzymatic glycosylation with clickable modified sugar donors followed by Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). The resulting array can be characterized by MALDI-TOF MS, to assess the extent of glycan modification
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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