Ieva Sutkeviciute

A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus

DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the μm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low μm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells.

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

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. &bgr; 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 &bgr; chemokines MIP-1&agr;, MIP-1&bgr; and RANTES. Conclusion:Dendron 12 thus inhibits HIV-1 infection by competition with binding of HIV to DC-SIGN and stimulation of &bgr;-chemokine production. Dendron 12 represents a promising lead compound for the development of anti-HIV topical microbicides.

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

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.

Structure of a Glycomimetic Ligand in the Carbohydrate Recognition Domain of C-type Lectin DC-SIGN. Structural Requirements for Selectivity and Ligand Design

In genital mucosa, different fates are described for HIV according to the subtype of dendritic cells (DCs) involved in its recognition. This notably depends on the C-type lectin receptor, langerin or DC-SIGN, involved in gp120 interaction. Langerin blocks HIV transmission by its internalization in specific organelles of Langerhans cells. On the contrary, DC-SIGN enhances HIV trans-infection of T lymphocytes. Thus, approaches aiming to inhibit DC-SIGN, without blocking langerin, represent attractive anti-HIV strategies. We previously demonstrated that dendrons bearing multiple copies of glycomimetic compounds were able to block DC-SIGN-dependent HIV infection in cervical explant models. Optimization of such ligand requires detailed characterization of its binding mode. In the present work, we determined the first high-resolution structure of a glycomimetic/DC-SIGN complex by X-ray crystallography. This glycomimetic, pseudo-1,2-mannobioside, shares shape and conformational properties with Manα1-2Man, its natural counterpart. However, it uses the binding epitope previously described for Lewis X, a ligand specific for DC-SIGN among the C-type lectin family. Thus, selectivity gain for DC-SIGN versus langerin is observed with pseudo-1,2-mannobioside as shown by surface plasmon resonance analysis. In parallel, ligand binding was also analyzed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol. These studies demonstrate that the complex, defined by X-ray crystallography, represents the unique binding mode of this ligand as opposed to the several binding orientations described for the natural ligand. This exclusive binding mode and its selective interaction properties position this glycomimetic as a good lead compound for rational improvement based on a structurally driven approach.

Selective Targeting of Dendritic Cell‐Specific Intercellular Adhesion Molecule‐3‐Grabbing Nonintegrin (DC‐SIGN) with Mannose‐Based Glycomimetics: Synthesis and Interaction Studies of Bis(benzylamide) Derivatives of a Pseudomannobioside

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and Langerin are C-type lectins of dendritic cells (DCs) that share a specificity for mannose and are involved in pathogen recognition. HIV is known to use DC-SIGN on DCs to facilitate transinfection of T-cells. Langerin, on the contrary, contributes to virus elimination; therefore, the inhibition of this latter receptor is undesired. Glycomimetic molecules targeting DC-SIGN have been reported as promising agents for the inhibition of viral infections and for the modulation of immune responses mediated by DC-SIGN. We show here for the first time that glycomimetics based on a mannose anchor can be tuned to selectively inhibit DC-SIGN over Langerin. Based on structural and binding studies of a mannobioside mimic previously described by us (2), a focused library of derivatives was designed. The optimized synthesis gave fast and efficient access to a group of bis(amides), decorated with an azide-terminated tether allowing further conjugation. SPR inhibition tests showed improvements over the parent pseudomannobioside by a factor of 3-4. A dimeric, macrocyclic structure (11) was also serendipitously obtained, which afforded a 30-fold gain over the starting compound (2). The same ligands were tested against Langerin and found to exhibit high selectivity towards DC-SIGN. Structural studies using saturation transfer difference NMR spectroscopy (STD-NMR) were performed to analyze the binding mode of one representative library member with DC-SIGN. Despite the overlap of some signals, it was established that the new ligand interacts with the protein in the same fashion as the parent pseudodisaccharide. The two aromatic amide moieties showed relatively high saturation in the STD spectrum, which suggests that the improved potency of the bis(amides) over the parent dimethyl ester can be attributed to lipophilic interactions between the aromatic groups of the ligand and the binding site of DC-SIGN.

Unique Dc-Sign Clustering Activity of a Small Glycomimetic: A Lesson for Ligand Design.

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.

Rational-Differential Design of Highly Specific Glycomimetic Ligands: Targeting DC-SIGN and Excluding Langerin Recognition

At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carbohydrate-based PAMPS or DAMPS (pathogen- or danger-associated molecular patterns, respectively) and promote immune response regulation. However, some CLRs are hijacked by viral and bacterial pathogens. Thus, the design of ligands able to target specifically one CLR, to either modulate an immune response or to inhibit a given infection mechanism, has great potential value in therapeutic design. A case study is the selective blocking of DC-SIGN, involved notably in HIV trans-infection of T lymphocytes, without interfering with langerin-mediated HIV clearance. This is a challenging task due to their overlapping carbohydrate specificity. Toward the rational design of DC-SIGN selective ligands, we performed a comparative affinity study between DC-SIGN and langerin with natural ligands. We found that GlcNAc is recognized by both CLRs; however, selective sulfation are shown to increase the selectivity in favor of langerin. With the combination of site-directed mutagenesis and X-ray structural analysis of the langerin/GlcNS6S complex, we highlighted that 6-sulfation of the carbohydrate ligand induced langerin specificity. Additionally, the K313 residue from langerin was identified as a critical feature of its binding site. Using a rational and a differential approach in the study of CLR binding sites, we designed, synthesized, and characterized a new glycomimetic, which is highly specific for DC-SIGN vs langerin. STD NMR, SPR, and ITC characterizations show that compound 7 conserved the overall binding mode of the natural disaccharide while possessing an improved affinity and a strict specificity for DC-SIGN.