Design and synthesis of inhibitors of DC-sign mediated infections

DC-SIGN (Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin) is a C-type (Calcium dependent) lectin, expressed as homotetramers (presenting four copies of a Carbohydrate Recognition Domain (CRD) at the C-terminus) on the surface of immature Dendritic Cells. [1] Dendritic Cells (DCs) areone of the most important class of Antigen Presenting Cells (APCs). They recognize many pathogens through various receptors such as DC-SIGN. After recognition, the pathogen is internalized and DCs mature and migrate to lymph nodes.[2] Then, DCs relay the corresponding processing antigens as MHC complexes to naive T-cells, which differentiate allowing the appropriate immuno-reponse. Some of these pathogens, such as HIV, hijack this mechanism to infect the immune system: they are recognized by DCs but escape the processing pathway. Thus, they can remain “hidden” inside the dendritic cells for many days, being able to reach and infect their target T-cells. The main carbohydrate ligand recognized by DC-SIGN is the high mannose glycan (Man)9(GlcNAc)2 , also known as Man9, a branched oligosaccharide which is presented in multiple copies by several pathogen glycoproteins (gp120, GP1, …). Hence, multivalent mannose display should be an adequate strategy to interact with this lectin with high affinity. In vivo, mannosides are normally hydrolyzed by mannosidases: the use of a structural mimic in place of the natural sugar could avoid an easy degradation in a biological environment. The aim of this project is to design and prepare products that meet these requirements. So far we have demonstrated that the monovalent mimic 1 shown in Figure 1[3] interacts with DC-SIGN (using NMR) and inhibits the DC-SIGN mediated infection in a pseudo-typed Ebola virus model. Moreover, this molecule has been conjugated to a Boltorn-type, leading to neo-glycoconjugates that inhibit the binding of DC-SIGN to gp120 (envelope protein of HIV). In this communication we will report the synthesis of new monovalent inhibitors and the results of their binding assays by SPR. We will show also the synthesis of some multivalent compounds. Acknowledgments. This work was supported by Azioni Integrate Italia-Spagna (IT074ABCCM). [1] T. B. H. Geijtenbeek, , Y. van Kook, et al., Cell 2000, 100, 575-585. [2] Y. van Kooyk, T. B. H. Geijtenbeek, Nat. Rev. Immunol. 2003, 3, 697-709. [3] José J. Reina, Sara Sattin, Donatella Invernizzi, Silvia Mari, Lorena Martínez-Prats, Georges Tabarani, Franck Fieschi, Rafael Delgado, Pedro M. Nieto, Javier Rojo, Anna Bernardi, ChemMedChem , 2007, 2(7),1030-1036

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

Nanotechnology and the Treatment of HIV Infection

Suboptimal adherence, toxicity, drug resistance and viral reservoirs make the lifelong treatment of HIV infection challenging. The emerging field of nanotechnology may play an important role in addressing these challenges by creating drugs that possess pharmacological advantages arising out of unique phenomena that occur at the “nano” scale. At these dimensions, particles have physicochemical properties that are distinct from those of bulk materials or single molecules or atoms. In this review, basic concepts and terms in nanotechnology are defined, and examples are provided of how nanopharmaceuticals such as nanocrystals, nanocapsules, nanoparticles, solid lipid nanoparticles, nanocarriers, micelles, liposomes and dendrimers have been investigated as potential anti-HIV therapies. Such drugs may, for example, be used to optimize the pharmacological characteristics of known antiretrovirals, deliver anti-HIV nucleic acids into infected cells or achieve targeted delivery of antivirals to the immune system, brain or latent reservoirs. Also, nanopharmaceuticals themselves may possess anti-HIV activity. However several hurdles remain, including toxicity, unwanted biological interactions and the difficulty and cost of large-scale synthesis of nanopharmaceuticals

DC-SIGN neck domain is a pH-sensor controlling oligomerization: SAXS and hydrodynamic studies of extracellular domain

DC-SIGN is a C-type lectin receptor of dendritic cells and is involved in the early stages of numerous infectious diseases. DC-SIGN is organized into a tetramer enabling multivalent interaction with pathogens. Once formed, the DC-SIGN-pathogen complex can be internalized into compartments of increasing acidity. We have studied the pH dependence of the oligomerization state and conformation of the entire extracellular domain and neck region. We present evidence for equilibrium between the monomeric and tetrameric states of the extracellular domain, which exhibits a marked dependence with respect to both pH and ionic strength. Using solution x-ray scattering we have obtained a molecular envelope of the extracellular domain in which a model has been built. Our results highlight the central role of the neck domain in the pH-sensitive control of the oligomerization state, in the extended conformation of the protein, and in carbohydrate recognition domain organization and presentation. This work opens new insight into the molecular mechanism of ligand release and points to new avenues to block the first step of this important infection pathway

1,2-Mannobioside Mimic : synthesis, DC-SIGN Interaction by NMR and Docking, and Antiviral Activity

The design and preparation of carbohydrate ligands for DC-SIGN is a topic of high interest because of the role played by this Ctype 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 DCSIGN 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

Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation

HIV infection is pandemic in humans and is responsible for millions of deaths every year. The discovery of new cellular targets that can be used to prevent the infection process represents a new opportunity for developing more effective antiviral drugs. In this context, dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), a lectin expressed at the surface of immature dendritic cells and involved in the initial stages of HIV infection, is a promising therapeutic target. Herein we show the ability of a new tetravalent dendron containing four copies of a linear trimannoside mimic to inhibit the trans HIV infection process of CD4+ T lymphocytes at low micromolar range. This compound presents a high solubility in physiological media, a neglectable cytotoxicity, and a long-lasting effect and is based on carbohydrate-mimic units. Notably, the HIV antiviral activity is independent of viral tropism (X4 or R5). The formulation of this compound as a gel could allow its use as topical microbicide

Sedimentation Velocity Methods for the Characterization of Protein Heterogeneity and Protein Affinity Interactions

International audienceSedimentation velocity analytical ultracentrifugation is a powerful and versatile tool for the characterization of proteins and macromolecular complexes in solution. The direct modeling of the sedimentation process using modern computational strategies allows among others to assess the homogeneity/heterogeneity state of protein samples and to characterize protein associations. In this chapter, we will provide theoretical backgrounds and protocols to analyze the size distribution of protein samples and to determine the affinity of protein-protein hetero-associations