Publications by authors named "José J Reina"

32 Publications

Influence of the reducing-end anomeric configuration of the Man epitope on DC-SIGN recognition.

Org Biomol Chem 2020 Aug 30;18(31):6086-6094. Epub 2020 Jul 30.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Av. Américo Vespucio 49, Seville 41092, Spain.

High-mannose (ManGlcNAc) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man epitope conjugated to two terminal N-acetylglucosamines by otherwise rarely-encountered β-mannose glycosidic bond. Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to prepare high mannose. Herein, we report the synthesis of the Man epitope with both alpha and beta configurations at the reducing end, and subsequent evaluation of the impact of this configuration on binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results therefore indicate that the more synthetically-accesible Man alpha epitope may be deployed as ligand for DC-SIGN in both in vitro and in vivo biological assays.
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http://dx.doi.org/10.1039/d0ob01380cDOI Listing
August 2020

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes.

Chemistry 2020 Jun 21;26(34):7516-7536. Epub 2020 Apr 21.

Centro Singular de Investigación en Química Biolóxica e, Materiais Moleculares (CIQUS), Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782, Santiago de Compostela, Spain.

Tracking pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade, different small probes and supramolecular systems have emerged for in cellulo or in vivo pH tracking. However, pH reporting still presents critical limitations, such as background reduction, improved sensor stability, cell targeting, endosomal escape, near- and far-infrared ratiometric pH tracking and adaption to new imaging techniques (i.e., super-resolution). These challenges will require the combined efforts of synthetic and supramolecular chemistry working together to develop the next generation of smart materials that will resolve current limitations. Herein, recent advances in the synthesis of small fluorescent probes, together with new supramolecular functional systems employed for pH tracking, are described with an emphasis on ratiometric probes. The combination of organic synthesis and stimuli-responsive supramolecular functional materials will be essential to solve future challenges of pH tracking, such as improved signal to noise ratio, on target activation and microenvironment reporting.
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http://dx.doi.org/10.1002/chem.201904834DOI Listing
June 2020

Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses.

J Am Chem Soc 2019 09 11;141(38):15403-15412. Epub 2019 Sep 11.

Departamento de Química Orgánica, Facultad de Química , Universidad Complutense , 28040 Madrid , Spain.

After the last epidemic of the Zika virus (ZIKV) in Brazil that peaked in 2016, growing evidence has been demonstrated of the link between this teratogenic flavivirus and microcephaly cases. However, no vaccine or antiviral drug has been approved yet. ZIKV and Dengue viruses (DENV) entry to the host cell takes place through several receptors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), so that the blockade of this receptor through multivalent glycoconjugates supposes a promising biological target to inhibit the infection process. In order to get enhanced multivalency in biocompatible systems, tridecafullerenes appended with up to 360 1,2-mannobiosides have been synthesized using a strain-promoted cycloaddition of azides to alkynes (SPAAC) strategy. These systems have been tested against ZIKV and DENV infection, showing an outstanding activity in the picomolar range.
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http://dx.doi.org/10.1021/jacs.9b08003DOI Listing
September 2019

Glycosylated Cell-Penetrating Peptides (GCPPs).

Chembiochem 2019 06 12;20(11):1400-1409. Epub 2019 Apr 12.

Centro Singular de Investigación en Química Biolóxica e, Materiais Moleculares (CIQUS), Departamento de Química Orgánica, Universidade de Santiago de Compostela, Campus Vida, 15782, Santiago de Compostela, Spain.

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.
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http://dx.doi.org/10.1002/cbic.201800720DOI Listing
June 2019

Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes.

J Org Chem 2018 02 2;83(4):1727-1736. Epub 2018 Feb 2.

Departamento de Química Orgánica, Facultad de Química, Universidad Complutense de Madrid , E-28040 Madrid, Spain.

The synthesis of multivalent systems based on hexakis-adducts of [60]fullerene employing a biocompatible copper-free click chemistry strategy has been accomplished. A symmetric hexakis-adduct of fullerene bearing 12 maleimide units (3) is reported, and it has been employed to carry out the thiol-maleimide Michael addition. To achieve orthogonal click addition, an asymmetric derivative bearing one maleimide and 10 cyclooctynes has been synthesized. The sequential and one-pot transformations of the two clickable groups have been explored, finding the best results in the case of the one-pot experiment. This route has been used to obtain a biocompatible hexakis-adduct appended with two different biomolecules, carbohydrates, and amino acids.
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http://dx.doi.org/10.1021/acs.joc.7b02402DOI Listing
February 2018

Straightforward synthesis of Man, the relevant epitope of the high-mannose oligosaccharide.

Org Biomol Chem 2017 Oct;15(42):8877-8882

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Américo Vespucio 49, Sevilla, Spain.

The high-mannose oligosaccharide (or its corresponding Man epitope) is the most abundant structure present in pathogen envelope glycoproteins. These glycans play a key role in the pathogenesis of several pathogens and also in the communication with the immune system. Understanding the mechanism of action of these glycans requires the access to pure and chemically well-defined structures in reasonable amounts. The synthesis of these complex branched oligosaccharides is not trivial and few syntheses are reported in the literature with several synthetic and purification steps and low overall yields. In this work, we described a very efficient synthetic alternative to access this relevant Man epitope in a very straightforward manner.
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http://dx.doi.org/10.1039/c7ob02286gDOI Listing
October 2017

Topological Defects in Hyperbranched Glycopolymers Enhance Binding to Lectins.

Chemistry 2017 Nov 16;23(62):15790-15794. Epub 2017 Oct 16.

Departament de Química Analítica i Química Orgànica, Universitat Rovira i Virgili, C/ Marcel lí Domingo 1, 43007, Tarragona, Spain.

Central scaffold topology and carbohydrate density are important features in determining the binding mechanism and potency of synthetic multivalent of poly- versus monodisperse carbohydrate systems against a model plant toxin (Ricinus communis agglutinin (RCA )). Lower densities of protein receptors favour the use of heterogeneous, polydisperse glycoconjugate presentations, as determined by surface plasmon resonance and dynamic light scattering.
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http://dx.doi.org/10.1002/chem.201703432DOI Listing
November 2017

Glycodendrimers as Chondroitin Sulfate Mimetics: Synthesis and Binding to Growth Factor Midkine.

Chemistry 2017 Aug 3;23(47):11338-11345. Epub 2017 Aug 3.

Instituto de Investigaciones Químicas (IIQ), CSIC- Universidad de Sevilla, Américo Vespucio 49, 41092, Seville, Spain.

Chondroitin sulfate (CS) is a member of the glycosaminoglycan (GAG) family, a class of polysaccharides implicated in relevant biological functions. The structural complexity of these carbohydrates demands the development of simple glycomimetics as useful tools to study the biological processes in which GAGs are involved. In this work we described the synthesis of the disaccharide unit of the CS-E (GlcA-GalNAc(4,6-di-OSO )), in a multivalent presentation. Using a fluorescence polarization competition assay we have demonstrated that a hexavalent dendrimer of this disaccharide interact with midkine, in the low micromolar range. This result highlights the potency of these disaccharide-displaying multivalent systems as interesting mimetics of longer and synthetically more complex GAG oligosaccharides.
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http://dx.doi.org/10.1002/chem.201701890DOI Listing
August 2017

Cyclooctyne [60]fullerene hexakis adducts: a globular scaffold for copper-free click chemistry.

Chem Commun (Camb) 2016 Aug;52(69):10544-6

Departamento de Química Orgánica, Fac. CC. Químicas, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid, Spain. and IMDEA-Nanoscience, Campus Cantoblanco, 28049 Madrid, Spain.

The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications.
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http://dx.doi.org/10.1039/c6cc05484fDOI Listing
August 2016

Rapid and efficient synthesis of α(1-2)mannobiosides.

Org Biomol Chem 2016 Mar;14(10):2873-82

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Américo Vespucio, 49, 41092, Sevilla, Spain.

α(1,2)mannobiosides with different substituents at the reducing end have been synthesized by a common strategy using benzoyls as the permanent protecting groups and an acetyl as the orthogonal protecting group at position C2 of the glycosyl acceptor. The new synthetic strategy has been performed remarkably reducing the number of purification steps, the time of synthesis (less than 72 hours) and improving the overall yield at least three times with respect to the best procedure described in the literature at the moment. Additionally, this protecting group strategy is compatible with the presence of azido groups and the use of Cu catalyzed azide alkyne cycloaddition (CuAAC) also called "click chemistry" for conjugating the α(1-2)mannobiosides to different scaffolds for the preparation of mannosyl multivalent systems.
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http://dx.doi.org/10.1039/c6ob00083eDOI Listing
March 2016

Effective targeting of DC-SIGN by α-fucosylamide functionalized gold nanoparticles.

Bioconjug Chem 2014 Dec 24;25(12):2244-51. Epub 2014 Nov 24.

CNR-Institute of Molecular Science and Technologies (ISTM) , via Golgi 19, I-20133 Milan, Italy.

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-β-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.
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http://dx.doi.org/10.1021/bc500467uDOI Listing
December 2014

Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR.

Glycobiology 2014 Aug 14;24(8):766-78. Epub 2014 May 14.

Dipartimento di Chimica, Universita' degli Studi di Milano, via Golgi 19, 20133 Milano, Italy

Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individual's blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence.
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http://dx.doi.org/10.1093/glycob/cwu040DOI Listing
August 2014

Unique DC-SIGN clustering activity of a small glycomimetic: A lesson for ligand design.

ACS Chem Biol 2014 Jun 6;9(6):1377-85. Epub 2014 May 6.

Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) , Grenoble F-38027, France.

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development.
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http://dx.doi.org/10.1021/cb500054hDOI Listing
June 2014

Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.

Circulation 2012 Sep 9;126(12):1484-94. Epub 2012 Aug 9.

Neuroscience Department, University of Milan.

Background: The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury.

Methods And Results: Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL(-/-) mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats.

Conclusions: Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.112.103051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478764PMC
September 2012

Carbohydrate mimics and lectins: a source of new drugs and therapeutic opportunities.

Mini Rev Med Chem 2012 Dec;12(14):1434-42

Universita' degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, via Venezian 21, 20133, Milano, Italy.

Mimics of oligosaccharides capable of interfering with lectin activity are currently being pursued by a number of groups in an effort to produce tools for glycobiology and to design antagonists of medically relevant lectins. The field is reviewed in this chapter. After a brief overview of the state of the art, examples from our and others' studies on the dendritic cell receptor DC-SIGN are illustrated.
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http://dx.doi.org/10.2174/138955712803832690DOI Listing
December 2012

Both El Tor and classical cholera toxin bind blood group determinants.

Biochem Biophys Res Commun 2012 Feb 28;418(4):731-5. Epub 2012 Jan 28.

Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway.

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.
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http://dx.doi.org/10.1016/j.bbrc.2012.01.089DOI Listing
February 2012

A glycomimetic compound inhibits DC-SIGN-mediated HIV infection in cellular and cervical explant models.

AIDS 2012 Jan;26(2):127-37

Department of Clinical Sciences L. Sacco, University of Milan, Segrate, Italy.

Objective: Dendritic cell-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin (DC-SIGN) participates in the initial stages of sexually transmitted HIV-1 infection by recognizing highly mannosylated structures presented in multiple copies on HIV-1 gp120 and promoting virus dissemination. Inhibition of HIV interaction with DC-SIGN thus represents a potential therapeutic approach for viral entry inhibition at the mucosal level.

Design: Herein we evaluate the efficacy in inhibiting HIV-1 infection and the potential toxicity of a multimeric glycomimetic DC-SIGN ligand (Dendron 12).

Methods: The ability of Dendron 12 to block HIV-1 infection was assessed in cellular and human cervical explant models. Selectivity of Dendron 12 towards DC-SIGN and langerin was evaluated by surface plasmon resonance studies. β chemokine production following stimulation with Dendron 12 was also analyzed. Toxicity of the compound was evaluated in cellular and tissue models.

Results: Dendron 12 averted HIV-1 trans infection of CD4(+) T lymphocytes in presence of elevated viral loads and prevented HIV-1 infection of human cervical tissues, under conditions mimicking compromised epithelial integrity, by multiple clades of R5 and X4 tropic viruses. Treatment with Dendron 12 did not interfere with the activity of langerin and also significantly elicited the production of the β chemokines MIP-1α, MIP-1β and RANTES.

Conclusion: Dendron 12 thus inhibits HIV-1 infection by competition with binding of HIV to DC-SIGN and stimulation of β-chemokine production. Dendron 12 represents a promising lead compound for the development of anti-HIV topical microbicides.
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http://dx.doi.org/10.1097/QAD.0b013e32834e1567DOI Listing
January 2012

Insights into molecular recognition of Lewis(X) mimics by DC-SIGN using NMR and molecular modelling.

Org Biomol Chem 2011 Oct 10;9(22):7705-12. Epub 2011 Oct 10.

Glycosystems Laboratory, Instituto de Investigaciones Químicas, CSIC-US, AmericoVespucio, 49 41092 Sevilla, Spain.

In this work, we have studied in detail the binding of two α-fucosylamide-based mimics of Lewis(X) to DC-SIGN ECD (ECD = extracellular domain) using STD NMR and docking. We have concluded that the binding mode occurs mainly through the fucose moiety, in the same way as Lewis(X). Similarly to other mimics containing mannose or fucose previously studied, we have shown that both compounds bind to DC-SIGN ECD in a multimodal fashion. In this case, the main contact is the interaction of two hydroxyl groups one equatorial and the other one axial (O3 and O4) of the fucose with the Ca(2+) as Lewis(X) and similarly to mannose-containing mimics (in this case the interacting groups are both in the equatorial position). Finally, we have measured the K(D) of one mimic that was 0.4 mM. Competitive STD NMR experiments indicate that the aromatic moiety provides additional binding contacts that increase the affinity.
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http://dx.doi.org/10.1039/c1ob05938fDOI Listing
October 2011

Second generation of fucose-based DC-SIGN ligands: affinity improvement and specificity versus Langerin.

Org Biomol Chem 2011 Aug 7;9(16):5778-86. Epub 2011 Jul 7.

Universita' degli Studi di Milano, Dipartimento di Chimica Organica e Industriale and CISI, Milano, Italy.

DC-SIGN and Langerin are two C-type lectins involved in the initial steps of HIV infections: the former acts as a viral attachment factor and facilitates viral invasion of the immune system, the latter has a protective effect. Potential antiviral compounds targeted against DC-SIGN were synthesized using a common fucosylamide anchor. Their DC-SIGN affinity was tested by SPR and found to be similar to that of the natural ligand Lewis-X (Le(X)). The compounds were also found to be selective for DC-SIGN and to interact only weakly with Langerin. These molecules are potentially useful therapeutic tools against sexually transmitted HIV infection.
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http://dx.doi.org/10.1039/c1ob05573aDOI Listing
August 2011

HIV microbicides: state-of-the-art and new perspectives on the development of entry inhibitors.

Future Med Chem 2010 Jul;2(7):1141-59

Universita' degli Studi di Milano, Dipartimento di Chimica Organica e Industriale and CISI, via Venezian 21, 20133 Milano, Italy.

Since the discovery of HIV at the beginning of the 1980s, numerous efforts have been devoted to the search of an efficient vaccine. There are at least 25 drugs available for HIV treatment, but no cure is available. The observation that therapy for HIV disease is life long and that these drugs are associated with a number of side effects underlines the need for approaches aimed at preventing rather than treating infection. Additionally, the economic burden of treatment for the HIV infection occupies an increasing share of healthcare expenditure, making current practices likely to become difficult to sustain in the long run. Unfortunately, no effective vaccine for this disease is foreseeable in the near future. Microbicides could be an alternate way to build preventative approaches to HIV infection. Strategies based on preventing the virus from reaching its target cells seem to have some room for development and application. In this review we explore the state-of-the-art of available microbicides, focusing on HIV entry inhibitors. In addition, we discuss new compounds that show anti-HIV activity, which could be effective candidates.
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http://dx.doi.org/10.4155/fmc.10.203DOI Listing
July 2010

Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations.

Mol Divers 2011 Aug 26;15(3):751-7. Epub 2011 Jan 26.

Institute for Research in Biomedicine (IRB Barcelona), Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, 08028 Barcelona, Spain.

Oligoribonucleotide conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two, or four glucose and galactose residues at the 5'-end have been prepared using phosphoramidite chemistry. Carbohydrate-modified siRNA duplexes have similar inhibitory properties than unmodified RNA duplexes in HeLa cells transfected with oligofectamine. When HeLa cells were treated with siRNA carrying one, two, or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues, although the efficacy of the process is not enough for efficient RNA interference experiments.
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http://dx.doi.org/10.1007/s11030-011-9305-6DOI Listing
August 2011

Highly polar carbohydrates stack onto DNA duplexes via CH/π interactions.

J Am Chem Soc 2011 Feb 18;133(6):1909-16. Epub 2011 Jan 18.

Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

Carbohydrate-nucleic acid contacts are known to be a fundamental part of some drug-DNA recognition processes. Most of these interactions occur through the minor groove of DNA, such as in the calicheamicin or anthracycline families, or through both minor and major groove binders such as in the pluramycins. Here, we demonstrate that carbohydrate-DNA interactions are also possible through sugar capping of a DNA double helix. Highly polar mono- and disaccharides are capable of CH/π stacking onto the terminal DNA base pair of a duplex as shown by NMR spectroscopy. The energetics of the carbohydrate-DNA interactions vary depending on the stereochemistry, polarity, and contact surface of the sugar involved and also on the terminal base pair. These results reveal carbohydrate-DNA base stacking as a potential recognition motif to be used in drug design, supramolecular chemistry, or biobased nanomaterials.
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http://dx.doi.org/10.1021/ja108962jDOI Listing
February 2011

Synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-DNA conjugates with different carbohydrate presentation.

Bioconjug Chem 2010 Jul;21(7):1280-7

Unidad de Biofisica, Centro Mixto CSIC-UPV/EHU, Universidad del Pais Vasco, 48080 Bilbao, Spain.

Oligonucleotide conjugates carrying carbohydrates at the 5'-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. Cellular surface adsorption and cellular uptake of carbohydrate oligonucleotide antisense sequences were measured using flow cytometric analysis. Conjugates with the glucose moiety linked through long spacers (15 to 18 atom distances) were internalized better than those with short linkers (4 atom distance) and than DNA control strands without sugar modification. Conjugates with tetravalent presentation of glucose did not improve cell uptake.
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http://dx.doi.org/10.1021/bc100079nDOI Listing
July 2010

Saturation transfer difference (STD) NMR spectroscopy characterization of dual binding mode of a mannose disaccharide to DC-SIGN.

Chembiochem 2008 Sep;9(14):2225-7

Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Américo Vespucio 49, 41092 Seville, Spain.

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http://dx.doi.org/10.1002/cbic.200800361DOI Listing
September 2008

Experimental measurement of carbohydrate-aromatic stacking in water by using a dangling-ended DNA model system.

Chemistry 2008 ;14(26):7828-35

Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Sevilla, Spain.

Protein-carbohydrate recognition is of fundamental importance for a large number of biological processes; carbohydrate-aromatic stacking is a widespread, but poorly understood, structural motif in this recognition. We describe, for the first time, the measurement of carbohydrate-aromatic interactions from their contribution to the stability of a dangling-ended DNA model system. We observe clear differences in the energetics of the interactions of several monosaccharides with a benzene moiety depending on the number of hydroxy groups, the stereochemistry, and the presence of a methyl group in the pyranose ring. A fucose-benzene pair is the most stabilizing of the studied series (-0.4 Kcal mol(-1)) and this interaction can be placed in the same range as other more studied interactions with aromatic residues of proteins, such as Phe-Phe, Phe-Met, or Phe-His. The noncovalent forces involved seem to be dispersion forces and nonconventional hydrogen bonds, whereas hydrophobic effects do not seem to drive the interaction.
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http://dx.doi.org/10.1002/chem.200800335DOI Listing
October 2008

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin.

Org Biomol Chem 2008 Aug 5;6(15):2743-54. Epub 2008 Jun 5.

Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Universidad de Sevilla, Américo Vespucio 49, Seville, Spain.

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.
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http://dx.doi.org/10.1039/b802144aDOI Listing
August 2008

Isolation, characterization, and localization of AgaSGNH cDNA: a new SGNH-motif plant hydrolase specific to Agave americana L. leaf epidermis.

J Exp Bot 2007 3;58(11):2717-31. Epub 2007 Jul 3.

Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, E-29071 Málaga, Spain.

GDSL and SGNH hydrolases are lipases involved in a wide range of functions, behaving in many cases as bifunctional enzymes. In this work, the isolation and characterization of AgaSGNH, a cDNA encoding a member of the SGNH-hydrolase superfamily from young leaf epidermis of the monocot Agave americana L., is reported. The protein possesses a typical signal peptide at its N-terminus that allows its secretion to the epidermis cell wall, as verified by immunolocalization experiments. In addition, the AgaSGNH sequence contains a His-Leu-Gly-Ala-Glu (HLGAE) motif which is similar to that observed in other plant acyltransferases. Expression levels by northern blot and in situ localization of the corresponding mRNA, as well as the immunolocalization of the protein in Agave young leaves indicate that the protein is specifically present in the epidermal cells. The detailed study performed in different parts of the Agave leaf confirms two aspects: first, the expression of AgaSGNH is limited to the epidermis, and second, the maximum mRNA levels are found in the epidermis of the youngest zones of the leaf which are especially active in cutin biosynthesis. These levels dramatically decrease in the oldest zone of the leaf, where the presence of AgaSGNH mRNA is undetectable, and the biosynthesis of different cuticle components is severely reduced. These data could be compatible with the hypothesis that AgaSGNH could carry out both the hydrolysis and the transfer, from an activated acyl-CoA to a crescent cutin in Agave americana leaves and, therefore, be involved in the still unknown mechanism of plant cutin biosynthesis.
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http://dx.doi.org/10.1093/jxb/erm136DOI Listing
January 2008

1,2-Mannobioside mimic: synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity.

ChemMedChem 2007 Jul;2(7):1030-6

Departamento de Química Bioorgánica, Instituto de Investigaciones Químicas, CSIC, Américo Vespucio 49, 41092 Sevilla, Spain.

The design and preparation of carbohydrate ligands for DC-SIGN is a topic of high interest because of the role played by this C-type lectin in immunity and infection processes. The low chemical stability of carbohydrates against enzymatic hydrolysis by glycosylases has stimulated the search for new alternatives more stable in vivo. Herein, we present a good alternative for a DC-SIGN ligand based on a mannobioside mimic with a higher enzymatic stability than the corresponding disaccharide. NMR and docking studies have been performed to study the interaction of this mimic with DC-SIGN in solution demonstrating that this pseudomannobioside is a good ligand for this lectin. In vitro studies using an infection model with Ebola pseudotyped virus demonstrates that this compound presents an antiviral activity even better than the corresponding disaccharide and could be an interesting ligand to prepare multivalent systems with higher affinities for DC-SIGN with potential biomedical applications.
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http://dx.doi.org/10.1002/cmdc.200700047DOI Listing
July 2007

Mannose glycoconjugates functionalized at positions 1 and 6. Binding analysis to DC-SIGN using biosensors.

Bioconjug Chem 2007 May-Jun;18(3):963-9. Epub 2007 Mar 10.

Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Américo Vespucio 49, E-41092 Sevilla, Spain.

The design of glycoconjugates to allow the generation of multivalent ligands capable of interacting with the receptor DC-SIGN is a topic of high interest due to the role played by this lectin in pathogen infections. Mannose, a ligand of this lectin, could be conjugated at two different positions, 1 and 6, not implicated in the binding process. We have prepared mannose conjugates at these two positions with a long spacer to allow their attachment to a biosensor chip surface. Analysis of the interaction between these surfaces and the tetravalent extracellular domain (ECD) of DC-SIGN by SPR biosensor has demonstrated that both positions are available for this conjugation without affecting the protein binding process. These results emphasize the possibility to conjugate mannose at position 6, allowing the incorporation of hydrophobic groups at the anomeric position to interact with hydrophobic residues in the carbohydrate recognition domain of DC-SIGN, increasing binding affinities. This fact is relevant for the future design of new ligands and the corresponding multivalent systems for DC-SIGN.
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http://dx.doi.org/10.1021/bc060369zDOI Listing
August 2007

Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding.

FEBS Lett 2006 May 31;580(10):2402-8. Epub 2006 Mar 31.

Institut de Biologie Structurale, UMR 5075 CEA/CNRS/Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor of dendritic cells and is involved in the initial steps of numerous infectious diseases. Surface plasmon resonance has been used to study the affinity of a glycodendritic polymer with 32 mannoses, to DC-SIGN. This glycodendrimer binds to DC-SIGN surfaces in the submicromolar range. This binding depends on a clustered organization of DC-SIGN mimicking its natural organization as microdomain in the dendritic cells plasma membrane. Moreover, this compound inhibits DC-SIGN binding to the HIV glycoprotein gp120 with an IC50 in the micromolar range and therefore can be considered as a potential antiviral drug.
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http://dx.doi.org/10.1016/j.febslet.2006.03.061DOI Listing
May 2006
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