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1. INTRODUCTION
1.1 Dendritic cells
Dendritic cells (DC) are professional antigen presenting cells (APC) that operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T cells.1 DC are present in those tissues that are in contact with external environment, such as the skin (where there is a specialized DC type called the Langerhans cell) and the inner lining of the nose, lungs, stomach and intestine. Their journey starts in the bone marrow where stem cells differentiate and migrate as precursor DC into the blood. From there, immature DC seed the peripheral tissues where they monitor for invading pathogens, which they capture and process to antigenic fragments that present at their cell surface in association with major histocompatibility complex II (MHC-II). Upon pathogen capture, immature DC receive activation signals, which initiate their maturation and migration to secondary lymphoid organs in order to present the processed antigens to naïve T cells. Both maturation and migration of DC are carefully orchestrated by panoplies of chemokines and adhesion molecules; chemokines control the differentiation stages of the DC and direct the migration of the various DC subtypes.
Once in the T cell area of lymph nodes , chemokines attract naïve T cells toward the DC, enabling maximal exposure of the MHC-presented peptide repertoire to the T cells. Adhesion molecules are crucial for all cellular interactions of the DC during its journey from bone marrow into blood, from blood into peripheral tissues, and subsequently into lymphoid tissue. Adhesion receptor and costimulatory molecules enable the establishment of contact between naïve T cells and DC, providing sufficient stability to allow scanning of the MHC II-peptide complex and to induce T cell receptor triggering. Recently, many new cell-surface molecules have been identified on DC that may contribute to their function in controlling innating as well as adaptive immunity. In particular, a large diversity of C-type lectin has been identified on DC, including Langering, the mannose receptor MR and DEC-250: some of these regulate pathogen
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recognition while others regulate signalling or cellular interactions such as DC migration and T-cell binding.1 Lectins are carbohydrate-binding protein, macromolecules that are specific for sugar moieties. Lectins perform recognition on the cellular and molecular level and play a role in biological recognition phenomena involving cells, carbohydrate and proteins.
C-type lectins are characterized by a carbohydrate recognition domain (CRD) that interacts with proteins with either mannose or galactose side chains in a calcium-dependent manner. The C-type lectins on DCs have a mannose-type specificity, and binding of mannosilated ligands can be blocked by mannan. However, the number of CRDs present in these lectins differs and the complexity of the mannose groups that they recognize is distinct. Langerin contains a single CRD and functions as an endocytic receptor on skin epidermal Langerhans cells. Instead, the MR has eight potential CRDs, of which two were shown to be functional for binding end-standing mannose groups on a variety of pathogens and Ags, illustrating its role in innate immunity.2
1.2 DC-SIGN, a DC-specific C-type lectin
In 2000, Geijtenbeek et al. identified DC-SIGN, a novel DC-specific adhesion receptor on human DC, which is essential in several key functions throughout the life cycle of DC. DC-SIGN was discovered by the observation that DC bind the intercellular adhesion molecule ICAM-3 with very high affinity. ICAM-3 is a member of immunoglobulin superfamily, which is expressed at high levels on resting T cells and might be important in establishing the initial DC-T cell interactions2. Geijtenbeek and his group discovered that instead of the common ICAM-3 integrin receptors LFA-1 and αDβ2, the initial interaction between DC and resting T cells is integrin independent and requires Ca2+ . They designed this receptor DC –specific ICAM-3 grabbing nonintegrin (DC-SIGN). DC-SIGN binding to ICAM-3 is calcium dependent and DC-SIGN CRD binds two Ca2+ ions, one essential for the tertiary structure and the other for coordinating ligand binding. They observed that DC-SIGN-ICAM-3 interactions are transient and are of particular importance in the activation of resting T cells. They proposed a model in which the initial interaction of DC with resting T cells is mediated
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by DC-SIGN-ICAM-3, followed by interactions through other adhesion molecules such as LFA-1, that is involved in more stable DC-T cell contacts. The transient nature of DC-SIGN-ICAM-3 interactions enables DC to interact with a large number of resting T cells. Instead of the resting T cells, activated T cells express a lower amount of ICAM-3 and this observation suggests the importance of DC-SIGN-ICAM-3 interactions in primary immune responses rather than secondary responses. In situ DC-SIGN is exclusively expressed by DC subsets present in T cell area of tonsils, lymph nodes and spleen. Thus, DC-SIGN expression in situ correlates well with its function as an important mediator of DC-T cell clustering and T cell activation. In skin, DC-SIGN expression is found on dermal DC whereas it is not expressed by Langerhans cell in the epidermis. In mucosal tissues such as rectum, uterus and cervix, DC-SIGN is abundantly expressed by DC present in lamina propria, further substantiating the importance of the localization of DC as a first-line defense against viruses and pathogens.
Next to ICAM-3, DC-SIGN binds another adhesion molecule ICAM-2 ,expressed on the endothelium of blood and lymphatic vessels as well as on high endothelial vascular cells and leukocytes. ICAM-2 plays a central role in mediating leukocyte recirculation as well as homing into secondary lymphoid tissues. The two interactions exhibit similar features: indeed the enzymatic removal of the N-linked carbohydrates from ICAM-2 and ICAM-3 completely abrogates the binding of DC-SIGN. However DC-SIGN interacts with ICAM-2 differently from that with ICAM-3. The distinct carbohydrate structure and/or the different size of the ICAM molecules may determine the manner of interaction. Moreover, only the DC-SIGN-ICAM-2 interaction resists shear stresses encountered under physiological flow conditions, and cells tether to and roll on ICAM-2 surface.3
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1.3 DC-SIGN structure
DC-SIGN is a tetrameric type II transmembrane protein. Each subunit comprises a short, cytoplasmic N-terminal domain with several intracellular sorting motif, an extracellular stalk of seven and half repeats containing 23 amino acid residues, and a C-terminal carbohydrate recognition domain (CRD). The extracellular CRD is stabilized by an α-helical stalk, which specifically recognizes glycosylated proteins, and binds ligands bearing mannose and related sugars. Indeed, most C-type lectins expressed by DC have specificity for mannose-containing carbohydrate: however, each C-type lectin may recognize a unique branching and positioning of mannose residues on a given pathogen or cell-surface structure. For example, the MR recognizes branch-end mannose residues, whereas DC-SIGN recognizes high-mannose residues located more internally within a glycan structure1.
The formation of multimeric complexes or conformational changes of their receptor is a possible way of increasing binding affinity of ligands containing repetitive sugar moiety. DC-SIGN tetramerization is thought to have the main impact on binding affinity, and occurs through the DC-SIGN neck-repeat domain. The hydrophobic necks are believed to stabilize the DC-SIGN oligomers and project the CRDs away from the cell surface, in a favourable position for appropriate multivalent interaction with glycan ligands. The repeats of the neck form extended stalks, stabilized largely by lateral interactions of α-helical regions in the 23-amino-acid repeats. This helical neck shape presents hydrophobic residues in recurring intervals of 3-5 that stack spontaneously to form dimers or tetramers. The organization into tetramers also amplifies the specificity and defines the set of pathogens that are recognized by SIGN. In this way, DC-SIGN binds particularly well to closely spaced oligosaccharides on the envelopes of viruses and membranes of parasites.
However, some adjustments of this stringent binding model have been demonstrated very recently, showing a certain flexibility allowed for conformational changes in DC-SIGN upon ligand binding. This allows it to adapt to the arrangement of target monosaccharides and thus enables all CRDs to interact with their ligands. The multimeric organization and conformational flexibility of DC-SIGN molecules on the DC surface is therefore necessary for effective and selective binding of various mannose-containing oligosaccharide patterns3.
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a) DC-SIGN schematic structure b) DC-SIGN tetramer
Fig. 1.1 a) Schematic structure of DC-SIGN and its main subunits. b) DC-SIGN
tetramerization through hydrophobic residues stacking in the neck repeat domain.
Apart from the tetramerization, DC-SIGN forms clusters that organize in membrane microdomains.3-5 This organization on the plasma membrane is important for the binding and internalization, suggesting that clustered assemblies act as a functional docking site for pathogens (Figure 1.2).
Figure 1.2 Tetramerization of DC-SIGN and further clustering allows high binding avidity
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1.4 DC-SIGN as Ag receptor on DCs
Various pathways exist by which DCs internalize soluble Ags for presentation to T cells. Lectins that function as Ag receptors contain carbohydrate-binding motifs and facilitate binding and uptake of glycosylated ligands. As a C-type lectin, DC-SIGN also functions as an Ag receptor on human DCs that can endocytose soluble ligand into lysosomal compartments, resulting in processing of ligand and subsequent presentation to T cells. The dual role of DC-SIGN as adhesion receptor and Ag receptor provides DCs with a functional receptor throughout their life span. The two different functions do not necessarily affect each other, because Ag internalization through DC-SIGN does not affect ICAM-3 binding or T cell proliferation. In particular, a recent study demonstrate that DC-SIGN functions as endocytic receptor and is constitutively internalized from the cell surface upon ligand binding. The cytoplasmic tail of DC-SIGN contains several motifs that may direct its intracellular targeting: a tyrosine based motif (Y-based), a triacidic cluster (EEE) and a dileucine motif(LL).
Gejitenbeek2 at al. demonstrated that the dileucine motif supports internalization of DC-SIGN -ligand complexes from the cell surface in transfected cells, because mutation of this motif inhibits DC-SIGN internalization. Schlesinger et al. led a study in which they mutate each of this motifs by alanine substitution and tested their roles in phagocytosis and receptor-mediated endocytosis of the higly-mannosilated ligands,
Mycobacterium tuberculosis mannose-capped lipoarabinomannano (ManLAM) and
HIV-surface glycoprotein gp120. The EEE mutant of DC-SIGN showed a reduced cell surface expression, near abolishment in the phagocytosis of ManLAM-coated beads and a marked reduction in the endocytosis of soluble gp120. Although, the mutant Y of DC-SIGN did not exhibit any effect on phagocytosis and intracellular trafficking to the phagolysosome, the LL mutant caused the majority of the receptor and/or ligands to remain bound to the cell surface, indicating a role for the LL motif as an internalization signal.
Collectively, a dual role has been indicated for the EEE motif as a sorting signal in the secretory pathway and a lysosomal targeting signal in the endocytic pathway.4 Accordingly, DC-SIG- ligand complexes are targeted to late endosomal or lysosomal compartments where ligands are processed for MHC class II presentation to T cells, indicating an important role for DC-SIGN as an antigen receptor.
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1.5 DC-SIGN as a novel HIV-receptor
DCs efficiently capture HIV-1 through a high-affinity interaction of gp120 with DC-SIGN and mediate subsequent transmission of HIV-1 to T cells. DC-DC-SIGN does not function as a classical HIV-1 entry receptor, but acts as an HIV-1 trans receptor that binds HIV-1 and transmits it very efficiently to neighboring permissive target cells. DC-SIGN is capable of binding to the gp120 envelope protein of HIV-1, which is heavily glycosylated and contains high mannos- type oligosaccharides. Site-directed mutagenenesis demonstrated that DC-SIGN binding to both gp120 and ICAM-3 is mediated by Ca2+ and that the interaction with ICAM-2, ICAM-3 and gp120 is blocked by the polycarbohydrate mannan, suggesting that carbohydrates are involved. However, the binding of DC-SIGN to gp120 differs from that to ICAM-3; indeed, DC-SIGN interacts with enzymatically deglycosylated and nonglycosylated gp120. The polycarbohydrate mannan is able to inhibit ICAM-3 and gp120 binding to DC-SIGN by occupying the binding site of DC-SIGN, suggesting that the carbohydrate and protein interactions to DC-SIGN are mediated by overlapping but distinct binding sites. 1,3 DC-SIGN does not only capture and transmit HIV-1from mucosa to lymphoid tissues, but also enhances infection of T cells. Indeed, the binding of HIV-1 to DC-SIGN alone is not sufficient for transmission of HIV-1, as HIV-1 binding and transfer by DC-SIGN have been shown to be dissociated functions. The complex HIV-1-DC-SIGN is promptly internalized via clathrin-coated pits to DCs’ endosomes, where the acidic endosomal media cause ligand to dissociate from DC-SIGN. Free DC-SIGN is then recycled to the DC surface while bound ligand are lysed and processed. Indeed, a large part of the HIV-1 that enters the DCs is destroyed by this mechanism.
In contrast with these findings, HIV-1 bound to DC-SIGN is astonishingly stable and a small quantity of HIV-1 that enters DCs remains protected from the host immune system, while retaining its infectiveness. HIV-1 stays in DCs in a highly infectious state for days, hidden in multivesicular bodies that are different from endosomes and/or lysosomes. Thus, HIV-1-infected DCs are able to mediate transmission of the virus to T cells through the formation of a so-called infectious synapse. Hodges et al. demonstrated that DC-SIGN signalling is responsible for viral synapse formation between DCs and T cell. Thus, HIV-1 induced DC-SIGN signalling triggers two paradoxical conditions, inhibiting DC maturation while inducing formation of viral
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synapse. This process may be facilitated by the DC-SIGN function as an adhesion molecule for ICAM-3 at the surface at the CD4+ T cells. Recently, it has been demonstrated that HIV-1 protein Nef induces DC-SIGN up-regulation in HIV infected DCs and this markedly stimulates DC-T cell clustering, which facilitate HIV transmission and dissemination. The intracellular trafficking of DC-SIGN is affected by the Nef, which can interact with the cell sorting machinery to down regulate expression levels of CD4 and MHC class I and thus facilitate immune invasion. Thus, DC-SIGN dictates the mode of HIV infection, from DC infection and immune system modulation, to HIV-1 transmission and dissemination, and HIV-1 clearly benefits from DC-SIGN-mediated signalling. Inhibition of pathogen interaction with DC-SIGN is a plausible concept for new antiinfectives, preventing not only localized infection of DCs, but also pathogen dissemination.3
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1.6 The structural basis of DC-SIGN’s CRD domain
Carbohydrate recognition domain (CRD) is a compact globular structure responsible for selective binding of terminal units found in large carbohydrate.5 The unique structural hallmark of such a domain is that binds a carbohydrate by Ca2+ ion, which act as a bridge between the protein and the “core monosaccharide” , while other ligand carbohydrate units (if present) form structural and bonding complementarity with the CRD. Several amino acid residues of the CRD offer six coordinate bonds for Ca 2+ ion, and the carbohydrate donates two coordinate bonds with its hydroxyls, so that the Ca2+is octacoordinated. Distinct CRD amino acid residues form hydrogen bonds with other hydroxyl groups on the carbohydrate, directly or through water bridges. Changes in the amino acid residues that interact with the “core monosaccharide” modify the carbohydrate-binding specificity of the lectin, so that specific carbohydrate is recognised. The free energy of such interactions is relatively weak for carbohydrate unit, due to the high desolvatation penalties of numerous hydroxyls upon carbohydrate binding.
However, the C-type lectins has means to obtain high binding affinity and avidity for specific glycans by oligomerization of its domain, as already mentioned. The clustering of CRDs influences not only the avidity (Figure 1.2), but also the lectin selectivity, since each individual CRD can act independently to bind end mono- or oligosaccharide moiety. DC-SIGN has a highly regulated recognition of its ligands as it selectively binds glycans with terminal D-mannose and L-fucose expressed on a number of bacteria, viruses and fungi. However the mere presence of D-mannose and L-fucose does not assure binding selectivity itself and monosaccharide binding to DC-SIGN CRD is generally very weak. Thus, DC-SIGN CRD forms a one-to-one complex with terminal mono or oligosaccharides , which relies upon already mentioned octacoordination of Ca2+ ion in the binding site by the “core monosaccharide”.5b
For D-mannose residues, the equatorial 3- and 4- hydroxyls each form coordination bonds with the Ca2+ in the binding site common to all C-type lectins, but also offer hydrogen bonds with amino acids that serve as Ca2+ ligands. A crucial structural features of a mannose residue is an axial position of the 2-hydroxyl group; this allows tight surface complementarity of the core mannose with the binding site. Equatorial position of the hydroxyl group on C(2) would probably prevent this tight binding due to
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clash, so hexopyranose with equatorial 2-hydroxyl groups do not form strong interactions. To increase binding affinity, other saccharide units form additional interactions with the binding site, while binding specificity is based on spatial constraints.
Fig. 1.4 D-mannose and its crucial positions. Two conformations of L-fucose
The difference in the affinity between high-mannose and fucosylated oligosaccharide results from a different spatial arrangement of the mannose- and fucose-based ligands demonstrated in two crystal structures of fucose-based tetrasaccharides LNFPII and Man4 tetrasaccharide bound to DC-SIGN CRD. The Man3 mannose moiety of Man4
inclines the rest of the molecule towards Phe313 with high surface complementarity, while the fucose moiety makes hydrophobic contact with Val 351 and positions the second saccharide in a vertical manner away from the protein surface. The binding mode presented for Man4 tetrasaccharide in which Phe313 forms steric hindrance is a
prevalent one for a mannose-containing oligosaccharides. However Phe313 residue is rather flexible and allows two distinct binding modes, both including coordination of the Ca2+by one mannose residue. In one mode the Man3 inclines the rest of the molecule towards Phe313 with high surface complementarity, while in the second, the binding orientation of the mannose at the principal Ca2+ is reserved and creates different interactions between the terminal mannose and the region around the Phe313. Thus we conclude that the binding mode of a specific oligosaccharide does not depend exclusively on the “core monosaccharide” involved, but is highly sensitive to the substitution pattern and 3D structure of adjacent monosaccharides.
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1.7 Binding of monovalent DC-SIGN antagonists
Binding of DC-SIGN natural ligands depends upon the presence of an L-fucose or D-mannose hexopyranose unit. However, carbohydrate-binding proteins’s affinity is generally weak for monosaccharide unit, so CBPs usually form strong interactions by binding massive glycans that bear several hundred terminal carbohydrate units.
Carbohydrates, especially large multivalent ones, possess unattractive pharmacokinetic properties: they are rapidly digested in the gut in most cases, and even if they survive the gut metabolism obstacles, they are practically unable to passively diffuse through the enterocyte layer in the small intestine. Their disadvantages in terms of low activity and /or insufficient drug-like properties can be modified by the design of glycomimetics compounds, that mimic the bioactive function of carbohydrates but have far better drug-like properties. This concept has been successfully used in the design of monovalent SIGN antagonist, low-molecular weight molecules that can occupy only one DC-SIGN CRD at a time, so they incorporate mono- and oligosaccharide structures and their mimetics. The design of these glycomimetics can be structurally divided into three sections:
The choice of monosaccharide unit, The choice of glycosidic bond surrogate,
The choice of adjacent saccharides or structure that contribute to overall binding affinity.
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1.7.1. The choice of monosaccharide unit
The most extensive work regarding the choice of monosaccharide unit shows that L-fucose can be incorporated in glycomimetic surrogates of Lewis-X trisaccharide to obtain even better affinity than the native trisaccharide.6 DC-SIGN can bind branched fucosylated structures that bear terminal galactose residue, such as the Lewis antigens expressed at the surface of viruses and bacteria as glycoconjugates (Figure 1.6 A). Binding of fucose-containing oligosaccharide to DC-SIGN has been reported by several groups; all of the new molecules contain a terminal fucose residue and have the structure of Lewis epitopes. However α-fucosides are both enzymatically and chemically lable; thus α-glycosilamides seem the right choice because have the advantage of being chemically stable and essentially unknown in nature, they are therefor likely not to be recognized by hydrolytic enzymes.
Figure 1.6 Structures of real Lewis-X trisaccharide and is mimic 1.1.
In order to design a fucose-based DC-SIGN ligand, Timpano el al.6 selected to use an α-fucosilamide anchor and connected it to a galactose or galactose mimic (Figure 1.6B). Thus they avoided glycosidic bonds, and reproduced the basic 3D features of the Lewis-X trisaccharide. They have established a simple protocol for their synthesis based on DeShong’s methodologyfor the synthesis of α-glucopyranosilamides.7
The full Lewis-X mimic 1.1 was shown to inhibit DC-SIGN binding of mannosylated BSA in the upper micromolar range (IC50= 350 µM), but the second generation of analogous compounds
failed to give any significant improvement over 1.1. STD-NMR studies of 1.1 with DC-SIGN extra-cellular domain have shown that only fucose residue makes strong contact
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with the DC-SIGN CRD. A reasonable explanation for this observation might be the before mentioned binding mode of the L-fucose moiety: it positions the second saccharide in vertical manner away from the protein surface, and thus the rest of the molecule fails from tight interactions with protein.
The L-fucose monosaccharide has the higest affinity for DC-SIGN among monosaccharides, and L-fucose should be the logical choice as the “core monosaccharide” when designing DC-SIGN antagonists. On the contrary, D-mannose has received most of the attentions as the majority of mono- and polyvalent DC-SIGN antagonists incorporate D-mannose as the “core monosaccharide”.
Scheme 1.1 The choice of “core monosaccharide” of DC-SIGN antagonists
The main carbohydrate ligand recognized by DC-SIGN is the high mannose glycan (Man)9(GlcNAC)2 also represented as (Man)9 , a branched oligosaccharide presented in
multiple copies by several pathogen glycoproteins (gp120, GPI). Unfortunately, the complexity of high mannose structures makes them unlikely candidates for the preparation of multivalent systems with potential applications in biomedicine. However, high mannose presents in all its arms terminal disaccharide Manα1-2Man which is
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likely to be involved in high mannose recognition process. Very recently it has also been demonstrated that high-density arrays of unbranched Manα1-2Man-terminated oligosaccharides bind to DC-SIGN almost as efficiently as the entire Man9. This
suggests an important role of nonreducing end Manα1-2Man fragment of Man9 in
DC-SIGN recognition.8 For these reasons D-mannose is used as “core monosaccharides” in many DC-SIGN antagonists.
Pseudo-1,2-mannobioside 1.2 (Scheme 1.1) and its analogues contain D-mannose unit substituted at the anomeric position with conformationally constrained cyclohexanediol, which mimes Manα1-2Man.8
The NMR of DC-SIGN extracellular domain with an azido derivative of 1.2 shows that the compound makes close contact with the protein, which is in agreement with the binding mode of Man4 tetrasaccharide. The inhibitory
activity of 1.2 on Ebola virus entry into DC-SIGN expressing Jurkat cells was quite high (IC50=0.62 mM) and this was the first functional assay showing that DC-SIGN
antagonism with small molecules might be used to inhibit viral transfection mediated by DC-SIGN.
Another example of substituted D-mannose as the “core monosaccharide” might be found in the work of Mitchell9 et al.: they have synthesized a small library of 2-C-substitued branched D-mannose analogue, of which compound 1.3 exhibited a 48-fold stronger binding to DC-SIGN. An innovative approach was used by Garber10 et al.: taking D-mannose as the lead structure, they have concentrated on the spatial relationship of hydroxyls at positions 2,3 and 4 and concluded that reduced shikimik acid should enable the same spatial relationship of hydroxyls. They have synthetized 192 derivates of reduced shikimic acid and compound 1.4 was found to be the most potent hit of this focused library (Scheme 1.2).
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1.7.2 The choice of glycosidic bond surrogate
The metabolic instability of glycosidic bonds makes it inappropriate for design of drugs and hence requires an appropriate surrogate when designing mimetics of oligosaccharides. Numerous change in carbohydrate structures have been successful in the design of glycosidase inhibitors and exactly these structures could be used to modify glycosidic bond instability. However, unwanted inhibition of glycosidases may as well induce side effects of potential drugs, so careful choice of glycosidic bond surrogates has to made if only its stability is the ultimate goal. The α-glycosidic bond found in both L-fucose and D-mannose containing oligosaccharides that bind to DC-SIGN was successfully replaced by Timpano et all. by a stable α-fucosylamide structure (compound 1.1, Scheme 1.1) and shown not to affect binding affinity in a detrimental sense. The derivatives of reduced shikimic acid (compound 1.4, Scheme 1.1) have two features that influence glycosidic bond stability: first, they have a thioglycosidic bond, which is proven to be metabolically more stable towards glycosidases, and second, they are a constitute of shikimic acid which is a carbasugar. Carbasugars lack anomeric reactivity, which implies their metabolic stability towards glycosidases and glycotransferases. Although glycosidic bond surrogates were often not used in the design of DC-SIGN antagonists, the stability of glycosidic bonds has challenged when designing stable glycomimetics.
1.7.3 The choice of adjacent structures that contribute to overall binding affinity Monosaccharides moieties other than “core monosaccharide” in the structure of DC-SIGN oligosaccharide ligands form not only additional interaction with the binding site, but also influence the binding specificity with spatial constraints and point other monosaccharide units towards or away from the protein surface. Notable quality of the adjacent monosaccharide units is that they do not form the same interactions like the “core monosaccharide”, but instead form a network of H-bonds, possibly through water molecules. For example, Man4 tetrasaccharide makes contact with DC-SIGN through at
least two water molecules , while one stabilizes its binding conformation. Alternatively both “core” and adjacent monosaccharides make surface complementarity and hydrophobic interactions. In particular, it has been demonstrated that Val351 in
DC-19
SIGN creates a hydrophobic pocket that strongly interacts with the fuc-α-1,3/4-GlcNAc moiety of the Lewis antigens11. So, although highly polar in nature, the adjacent monosaccharide units contribute significantly to overall binding free energy also by hydrophobic interactions apart from being mere “linker” to other structures. This implies that altering hydrophilic character of adjacent monosaccharide units to more hydrophobic surrogates should increase the free binding energy by increasing hydrophobic interactions. With this in mind, Timpano6 et all. have used the (1S, 1R)-2-aminocyclohexanecarboxylic acid as a scaffold/linker to attach D-galactose mimetic into the structure of compound 1.1 and its derivatives. The molecule was carefully chosen to mimic Lewis-X structure , by performing a conformational search of the candidates and overlapping the resulting conformations within 3 kcal mol-1 to the Lewis-X trisaccharide. The galactose mimetic was incorporated basing on the observation that galactose residue makes contact with the DC-SIGN CRD surface and is thus important for the bindng. Furthermore, the use of galactose mimic rather than galactose itself appeared to improve the structural similarity between the mimic and the Lewis-X structure by reducing the H-bonding interactions between the sugar and linker that distorted the ligand away from the desired shape6.
Scheme 1.3 (1S,2R)-2-aminocyclohexanecarboxylic acid as a scaffold/linker to attach
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In the series of mannose-based DC-SIGN antagonists, groups of Rojo and Bernardi have incorporated (1S,2S,4S,5S)-dimethyl-4,5-dihydroxycyclohexane-1,2-dicarboxylate as adjacent monosaccharide mimicking trans conformation of 1,2-hydroxyls in D-mannose (Scheme 1.4, compounds 1.2 and 1.5). The rational of this change is to imitate the 3D relationship of key hydroxyl in D-mannose moiety, while lowering the overall hydrophilicity. Furthermore, the cyclohexane saccharide mimics lack glycosidic bond and is thus metabolically stable.
Scheme 1.4 (1S,2S,4S,5S)-dimethyl-4,5-dihydroxycyclohexane-1,2-dicarboxylate as central monosaccharide surrogate that mimics “trans” conformation of 1,2-hydroxyls in D-mannose
Starting from compound 1.2 or its azido derivative, the group of Bernardi continued to pursue the idea of increasing the binding affinity by identifying two binding areas around Phe313 in the DC-SIGN binding site that were only partially occupied by cocrystallized tetramannoside Man4. These hydrophobic areas were targeted by attaching different hydrophobic moieties to deprotected carboxylates of pseudo-1,2-mannobioside 1.2. A number of mannose-based DC-SIGN antagonist were synthesized and the majority of them inhibited DC adhesion at low micromolar concentrations improving the potency of the starting compound 1.2 by two orders of magnitude (Scheme 1.5). Probably the same hydrophobic areas have contributed to the affinity of compound 1.4.
21 Scheme 1.5Increasing potency of DC-SIGN antagonists by attaching hydrophobic moieties to
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1.8 Pseudo-1,2-mannobiosides: an adequate strategy on DC-SIGN
antagonism
As already seen, one of the most successful strategy adopted in synthesizing DC-SIGN antagonists is the one in which a D-mannose “core monosaccharide” is connected by a glycosidic bond surrogate to a conformationally locked diol, which acts as a mimic of the reducing end mannose ring. Molecules with such properties are defined
“glycomimetics”, compounds that are lacking standard glycosidic linkages but resemble
a carbohydrate and can mimic or inhibits its function.
An important class of glycomimetics is represented by “carbasugars”. A carbasugar is a carbocyclic analogue of a true sugar in which the ring oxygen has been replaced by a metilene group (-CH2-). The term is also referred to any carbohydrate derivative or
other compound that has multiple hydroxyl groups and thus resembles a sugar or a saccharide. On the other hand, the structural substitution of the endocyclic oxygen atom with a methylene group leads to compounds more stable toward endogenous degradative enzyme. During the past few years a number of publications and reviews had illustrated the potential of sugar mimics in different field of drug discovery. The main advantage of this approach is rooted in the improved drug-like character of glycomimetics compared to natural carbohydrates. Sugar mimics are generally more soluble and membrane penetrant, less hydrophilic and less metabolically labile than the sugar themselves.12
Bernardi13 et al. in 2001 led a study in which they obtained a set of molecular scaffolds for the synthesis of glycomimetics, basing on the stereoselective synthesis of conformationally constrained cyclohexanediols. They observed that carbocyclic diols of the appropriate configuration can be used as substitutes of branching units in complex oligosaccharides. These portions do not bind directly the receptors, but act as a scaffold that orients the binding determinants in the appropriate conformation and provides a connection to the aglycons. For istance, the 3,4-disubstitued N-acetyl glucosamine (GlcNAc) of the LewisX tetrasaccharide, a residue that does not contain any critical groups for binding the LewisX target, has been successfully replaced using 1,2-trans-cyclohexanediol in the synthesis of a highly effective mimics. However, the use of simple cyclohexanediols is limited by two factors. First, the 1,2-cyclohexanediols are conformationally flexible. Hence, they cannot be used to replace those branching motifs that incorporate one or more axial substituents, and that are frequently
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encountered in bioactive oligosaccharides. Second, in biological systems the bioactivity of oligosaccharides is often expressed by multivalent clusters.
They concluded that the stereoisomeric 1,2-trans-dicarboxy-4,5-cyclohexanediols (DCCHDs) 1.7-1.9 (Fig. 1.5) overcome both the above-mentioned drawbacks and thus appear a very attractive alternative as mimics of scaffold monosaccharide.
Indeed, DCCHDs are highly conformationally stable thanks to the 1,2-trans-dicarboxy substitution that acts as a conformational lock for the cyclohexane ring. The Scheme 1.6 shows that for all isomers the chair featuring two equatorial carboxy groups is at least 2.7 kcal/mol more stable than the isomeric one, diaxially substituted. Accordingly, the diol functionality is also locked in the lowest energy conformation shown in Scheme 1.6 and can be used to reproduce vicinally disubstitued monosaccharides of any relative configuration. In fact, diol 1.7 recently succeeded in mimicking the cis diol of the 3,4-disubstitued galactose residue in the GM1 oligosaccharide and the resulting pseudo-GM1-molecule was shown to replicate the three-dimentional structure and cholera toxin –binding activity of its natural model. In addition, the carboxy groups of DCCHDs could be exploited for conjugation to various supports, thus allowing the synthesis of polyvalent pseudo-glycoconjugates.
Fig. 1.5 Conformationally
constrained dicarboxy cyclohexanediols 1.7-1.9 and their common precursor 1.10.
Scheme 1.6: Conformational equilibria of
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Due to this finding and aimed from the increasing interest on DC-SIGN, the group tried to applied their discovery to the synthesis of DC-SIGN antagonist. They synthetizes a set of pseudo-1,2-mannobiosides in which the reducing end mannose ring is substituted by a conformational locked diol with a short tether by either an azido 1.11b or an amino
1.11a group (Fig. 1.7). This structure is good candidate to generate new multivalent
DC-SIGN ligands that may display enhanced metabolic stability.
Fig. 1.7 Chemical structure of pseudo-1,2-mannobioside 1.11, Manα1-2Man 1.12 and
numbering of the possible Man fragments.
1.8.1. Synthesis of Pseudo-1,2-mannobiosides
The pseudo-1,2-α-mannobioside 1.11, designed to interact with DC-SIGN, was prepared by connecting an appropriately protected mannose unit 1.13 (the glycosyl donor) to a conformationally restricted dimethyl cyclohexanedicarboxilate 1.14 (the glycosyl acceptor) which acted as a mimic of the reducing end mannose unit, bearing an ethoxy chain at C1 with an azido or amino functionality at the end.
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The synthesis of the glycosyl acceptor starts from the epoxide 1.15 (Scheme 1.9), which is the intermediate of the previously mentioned stereoselective synthesis of DCCHDs. In particularly, the desired glycosyl acceptor 1.14 derives from the procedure that lead to a trans-diaxial diol starting from a common precursor 1.16 (Scheme 1.6).14
Scheme 1.6 1,2-dicarboxy-cyclohexene, precursor of the desired glycosyl acceptor
The 1,2-dicarboxy-4-cyclohexene 1.16 can be synthesized in both enentiomeric forms following a facile, large scale synthesis protocol. The synthesis starts from the Bolm’s desymmetrization of anhydrides. Treatment of the commercially available tetrahydrophthalic anhydride 1.16’ with MeOH in the presence of 1.1 equiv of quinine or its quasienantiomer quinidine results in the highly stereoselective formation of the (1S,2R)-monoacid 1.16’a (ee 90%) or the (1R,2S) enantiomer 1.16’b (ee 93%) respectively.
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Scheme 1.7 Bolm’s desymmetrization of tetrahydrophthalic anhydride.
Starting from 1.16’a, the (1S,2S)-trans-diacid 1.16 was obtained by the sequence depicted in Scheme 1.8. The t-BuOK epimerization gave the 4:1 trans:cis mixture, which was directly hydrolyzed to give the desired diacid 1.16, contaminated by a 20% of the cis isomer. The latter was selectively transformed in the starting hydrophthalic anhydride by treating the crude hydrolysis product with 0.2 equiv of Ac2O under
benzene reflux. Under these conditions the trans diacid 1.16 was left unchanged and could be isolated in 80% yield by crystallization from benzene.
Scheme 1.8 Stereoselective synthesis of (1S,2S)-trans-cyclohex-4-ene-1,2-dicarboxylic
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The 1,2-dicarboxycyclohexene was subjected to a Fischer esterification in MeOH. The oxidation with MCPBA of the bis dimethyl ester afforded the epoxide 1.1513 , which was is subjected to alcoholysis with 2-bromoethanol, promoted by Cu(OTf)2 as Lewis
acid catalyst.14 The cyclohexane derivative was subjected to an SN2 at the
bromoethan-substituent, which gave the desired glycosyl acceptor 1.14.
Scheme 1.9Synthesis of the glycosyl acceptor
The synthesis of the glycosyl donor,15 trichloroacetimidate TCA 1.13 started from commercially available (+)-D-mannose, which was initially subjected to acetylation to yield the fully O-acetyl derivative, the penta-O-acetyl-mannopyranose 1.13a. The D-mannopyranose was elaborated to 2,3,4,6-tetra-O-acetyl-D-D-mannopyranose 1.13b by chemoselective removal of anomeric acetyl group by hydrazine acetate. Subsequent treatment of the resulting hemiacetal with a large excess of trichloroacetonitrile in presence of a catalytic amount of DBU gave the only thermodinamically more stable α-trichloroacetimidate 1.13 in excellent yield.
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The pseudodisaccharide 1.17 was assembled in 50% yield by glycosylation of the acceptor 1.14 with the tetracetylmannose trichloroacetimidate 1.13. The reaction was promoted by 0.2 equiv of TMSOTf in CH2Cl2 at -20°C. Deacetylation with MeONa in MeOH afforded the pseudodisaccharide 1.11b, which is the first derivative that could been used as DCSIGN antagonist. Azide reduction using hydrogen at one atmosphere and Pd on carbon as catalyst, afforded the desired compound 1.11a.8
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1.8.2 NMR, Docking and infection studies.
NMR and docking studies have been performed to study the interaction of the 1,2-mannobioside mimics obtained with DC-SIGN, demonstrating that 1.11a and 1.11b are good ligands for the target. The interaction of 1.11b with DC-SIGN extracellular domain (EC) was studied in solution by NMR spectroscopy. Addition of DC-SIGN EC domain to a solution of 1.11b induced a broadening of the resonance signals in the 1H NMR spectrum, indicating that binding occurs. To observe the binding event, Bernardi’s group used STD and TR-NOESY experiment, by which can be evaluate the ligand exchanging between free and bound state. Both experiments confirmed that binding occurs and indicated that the ligand is in close contact with the protein. Furthermore, the two limit conformations E(extended) and S (stacked) of 1.11b are both present in the TR-NOESY spectrum, suggesting that both of them may be able to interact with the protein. However, some spin diffusion noise in the spectrum does not allow definitive conclusions about the preferred conformation of the ligand in the binding site.
Docking studies were performed using PDB structure of the DC-SIGN Man4 complex
as a model of DC-SIGN-1.11b complex and QM-Polarized ligand docking protocol. In this protocol ligands are docked with Gilde, then charges on the ligand induced by the receptor are calculated by quantum mechanical method and set of best ligand pose are redocked. The methyl ether glycoside 1.11c (Figure 1.6) was used as computational model of 1.11b. All the complexes obtained appeared to maintain the interactions between the Ca2+ atom and two hydroxyl groups of the nonreducing end mannose unit. The antiviral activity of 1.11a was tested using an infection model based on Ebola envelope-pseudotyped viruses and Jurkat cells expressing DC-SIGN. The activity of
1.11a in this infection model was compared with the natural disaccharide derivative 1.12a (Fig.1.8) presenting the same linker at the anomeric position. Different
concentrations of 1.11a and 1.12a were used and assays were repeated at least three times for each concentrations.
The results showed that the IC50 measured for the mimic 1.11a was approximately three
times lower than the value estimated for the corresponding disaccharide 1.12a. Then, the designed pseudodisaccharide 1.11a is a stronger inhibitor than the corresponding
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disaccharide 1.12a and could be considered as a promising candidate to prepare multivalent systems to be used as inhibitors of viral infection.
