key: cord-1036845-n9uaibad authors: Ernst, Beat; Magnani, John L. title: From carbohydrate leads to glycomimetic drugs date: 2009-07-24 journal: Nat Rev Drug Discov DOI: 10.1038/nrd2852 sha: bbd856312d3bd0b1812fdd8860854d09d800c093 doc_id: 1036845 cord_uid: n9uaibad Carbohydrates are the most abundant natural products. Besides their role in metabolism and as structural building blocks, they are fundamental constituents of every cell surface, where they are involved in vital cellular recognition processes. Carbohydrates are a relatively untapped source of new drugs and therefore offer exciting new therapeutic opportunities. Advances in the functional understanding of carbohydrate–protein interactions have enabled the development of a new class of small-molecule drugs, known as glycomimetics. These compounds mimic the bioactive function of carbohydrates and address the drawbacks of carbohydrate leads, namely their low activity and insufficient drug-like properties. Here, we examine examples of approved carbohydrate-derived drugs, discuss the potential of carbohydrate-binding proteins as new drug targets (focusing on the lectin families) and consider ways to overcome the challenges of developing this unique class of novel therapeutics. glycanbinding epitopes. These discoveries have led to a renaissance in glycobiology. They also provide a con tinuous supply of carbohydraterelated targets for the structurebased design of new chemical entities that mimic bioactive carbohydrates, and form a novel class of therapeutics. Carbohydrate and carbohydrate-derived drugs Although carbohydrates play an important part in a vast array of biological processes, carbohydrate and carbo hydratederived drugs cover only a limited area of the world of therapeutics (FIG. 1) . Many pathophysiologically important carbohydrate-protein interactions have yet to be exploited as a source of new drug targets. One reason might be the pharmacokinetic drawbacks that are inher ently linked to carbohydrates. As a result of their high polarity, they are unable to cross passively through the enterocyte layer in the small intestine -a prerequisite for oral availability. In addition, once systemically avail able by parenteral administration, carbohydrates suffer from fast renal excretion. When interactions with blood plasma components are possible, the plasma halflife that is required for a successful therapeutic application can be achieved. Prominent examples are the lowmolecularweight heparins, derived from animal tissue, and fondaparinux 3 (Arixtra; GlaxoSmithKline), which are used as anti coagulants. In other cases -such as the inhibition of The centre of chirality of a glycan that is generated by hemiacetal ring closure. From carbohydrate leads to glycomimetic drugs Beat Ernst* and John L. Magnani ‡ Abstract | Carbohydrates are the most abundant natural products. Besides their role in metabolism and as structural building blocks, they are fundamental constituents of every cell surface, where they are involved in vital cellular recognition processes. Carbohydrates are a relatively untapped source of new drugs and therefore offer exciting new therapeutic opportunities. Advances in the functional understanding of carbohydrate-protein interactions have enabled the development of a new class of small-molecule drugs, known as glycomimetics. These compounds mimic the bioactive function of carbohydrates and address the drawbacks of carbohydrate leads, namely their low activity and insufficient drug-like properties. Here, we examine examples of approved carbohydrate-derived drugs, discuss the potential of carbohydrate-binding proteins as new drug targets (focusing on the lectin families) and consider ways to overcome the challenges of developing this unique class of novel therapeutics. Nature Reviews | Drug Discovery Zanamivir (Relenza) 7 Oseltamivir phosphate (Tamiflu) 9 Miglustat (Zavesca) 226 Topiramate (Topamax) 227 Voglibose (Glustat) 4 Miglitol (Glyset) 5 Acarbose (Glucobay) 6 Fondaparinux (Arixtra) 10 n = 3-20; R = H or SO 3 Na; R 1 = SO 3 Na or Ac R 2 = H, R 3 = CO 2 Na or R 2 = CO 2 Na, R 3 = H Dalteparin sodium (Fragmin) 229 Sodium hyaluronate (Orthovisc) 228 Ardeparin sodium (Normiflo) 229 R = H or SO 3 (1/2Ca); R 1 = H, SO 3 (1/2Ca) or Ac R 2 = H, R 3 = CO 2 (1/2Ca) or R 2 = CO 2 (1/2Ca), R 3 A long polysaccharide chain consisting of repeating sulphated dissacharide units. αglycosidases in the brush border of the small intes tine for the treatment of diabetes (by voglibose 4 (Basen/ Glustat/volix; Takeda), miglitol 5 (Glyset; Pfizer) and acarbose 6 (Glucobay/Prandase/Precose; Bayer)) or the inhibition of viral neuraminidases in the pharyngeal mucosa (by zanamivir 7 (Relenza; GlaxoSmithKline))oral availability is not required. The paradigm of a glycomimetic drug in the classical sense is oseltamivir (Tamiflu; Gilead/Roche). Starting from a carbohydrate lead, drug likeness was achieved by systematically eliminating polar groups and meta bolic 'soft spots' 8 that were not required for affinity. Finally, by designing a prodrug, oral availability became possible 9 . Carbohydratebinding proteins are broadly classified into lectins 10 and sulphated glycosaminoglycan (SGAG) binding proteins 11, 12 . There are two categories of lectins present in vertebrates: the families of intra cellular lectins (for example, calnexin, ltype and Ptype lectins), which bind core oligosaccharide structures and are involved in glycoprotein processing and quality control, and the families of extracellular lectins (for example, galectins, Ctype, Itype and Rtype lectins), which recog nize terminal carbohydrate epitopes of other cells and pathogens. Extracellular lectins account for most of the molecular targets that are being investigated in current drug discovery programmes. Structures of currently approved drugs (trade name in brackets). These include glycosidase inhibitors that prevent the digestion of carbohydrates for the treatment of diabetes (voglibose 4 , miglitol 5 and acarbose 6 ) and the prevention of influenza virus infections (zanamivir 7 and oseltamivir 9 ); and sulphated glycosaminoglycans, which function as anticoagulants by binding to antithrombin III for the treatment of thrombosis (fondaparinux 3 , dalteparin 161 , ardeparin 161 , nardoparin 161 and enoxaparin 161 ). In addition, carbohydrate-derived drugs are used to treat Gaucher's disease (miglustat 162 ), epilepsy (topiramate 163 ) and osteoarthritis (sodium hyaluronate 164 ). The three-dimensional domain in a lectin that binds carbohydrate. A common three-dimensional structural domain shared by the carbohydrate structures Lewis a (Galβ (1) (2) (3) [Fuc(α1-4)] GlcNAc) and Lewis x (Galβ (1) (2) (3) (4) [Fuc(α1-3)]GlcNAc). By contrast, SGAGbinding proteins are heterogen eous and difficult to classify 11, 12 . Their ability to recognize SGAGs arises from clusters of cationic amino acids on unrelated proteins that confer the ability to recognize anionic structural motifs in extended SGAG chains. Typically, various SGAGbinding proteins interact with each SGAG with different affinities, and only a few SGAG sequences are exclusively recognized by a single SGAG binding protein. Here, we present the most promising drug candidates from the lectin families: selectins and dendritic cell specific ICAM3grabbing nonintegrin 1 (DCSIGn; also known as CD209) from the Ctype lectin family, myelin associated glycoprotein (MAG; also known as sialic acid binding immunoglobulinlike lectin 4A (Siglec 4A)) as an example of an Itype lectin, and PAI galactophilic lectin (PAIl), fucosebinding lectin PAIIl and minor component of type 1 fimbriae (FimH) as representatives of bacterial lectins. The hallmark of Ctype lectins is the involvement of Ca 2+ in the binding of glycans to their carbohydrate recognition domain (CRD). They have a wide range of biological func tions, such as intercellular adhesion, serum glycoprotein removal and pathogen recognition. Selectins. These are perhaps the most intensely studied mammalian carbohydratebinding proteins. First dis covered in 1989 , their functions as adhesion molecules are well understood 16 . The family consists of three members: Eselectin (also known as CD62E), Pselectin (also known as CD62P) and lselectin (also known as CD62l). They are composed of a Ca 2+ dependent CRD, an epidermal growth factor (EGF) domain, various short complementlike consensus repeats, a single transmembrane domain and an intracell ular tail. Although carbohydrates bind to a receptor site within the CRD, the neighbouring EGF domain influences binding affinity and specificity 17 . The three selectins have overlapping and distinct expression patterns, both temporally and spatially. Eselectin is expressed on endothelial cells by de novo pro tein synthesis 2-4 hours after stimulation by inflammatory mediators, such as interleukin 1β and tumour necrosis factorα. Pselectin is expressed on activated platelets and is also stored in Weibel-Palade bodies in endothelial cells, which fuse to the cell surface on activation, leading to the expression of Pselectin within minutes. lselectin is con stitutively expressed by most leukocytes and plays a major part in homing and trafficking of lymphocytes through the blood and lymphatic systems. All three selectins bind a common carbohydrate domain shared by sialyl Le a/x (sialyl lewis a (sle a ) and sialyl lewis x (sle x )) 18 . Interestingly, both of these carbohydrate sequences were originally discovered as cancerassociated antigens [19] [20] [21] and are prognostic indicators of metastatic dis ease 22 . Tumour cells coated with these carbohydrate chains are recognized as migrating leukocytes, allowing them to escape the bloodstream and metastasize to other organs and tissues, such as the lymph nodes and bone marrow 23, 24 . To functionally bind sialyl le a/x in vivo, both P and lselectins require additional interactions with negatively charged sulphate groups, either on the carbohydrate chain itself or on an adjacent peptide sequence. Eselectin has no such requirement and can functionally bind sialyl le a/x in glycolipids 25 and glycoproteins 26 . The involvement of negatively charged groups, such as sulphates and carboxylates, in the binding of l and Pselectin has led to one of the major pitfalls in designing smallmolecule inhibitors for the selectins. A wide range of structurally diverse, negatively charged molecules has been reported to bind P and lselectins. These include sulphatides 27 , heparins 28 , fucoidan 29 , sulphated dextran 30 , chondroitin sulphate 31 , dermatan sulphate 32 , tyrosine sulphates 33 , sulphated hyaluronic acid 34 and sulpho galabiose 35 . Such a range of molecules suggests that their inhibitory activity is due to nonspecific negativecharge interactions. In fact, a cautionary publication 36 described potent Pselectin activity found in trace contaminants of polyanions from ion exchange media used in the prepa ration samples. Thus, the specificity of smallmolecule, highly charged selectin antagonists that inhibit P and l but not Eselectin must be carefully evaluated. In diseases in which cell adhesion, extravasation of cells from the bloodstream or the migration of specific lymphocytes has been implicated in the pathology, selectins present an attractive therapeutic target. For example, E and Pselectins have been shown to mediate the acute adhesion and aggregation of leukocytes and erythrocytes during a vasoocclusive crisis in a mouse model of sickle cell disease 37, 38 . Furthermore, aberrant extravasation of cells from the bloodstream is the hall mark of many inflammatory diseases (such as asthma, colitis, arthritis and psoriasis) and cancer. Tumour cells that extravasate out of the bloodstream use the selec tin pathway to metastasize. Many solid tumours and adeno carcinomas, such as gastrointestinal 39 , pancreatic 40 , breast 41 , lung 42 and prostate 43 cancers, express high levels of sle x and sle a . Expression of these selectin ligands on the tumour cells of patients with gastric and colon cancers 44 is significantly correlated with poor survival 22 . Cimetidine (Tagamet; GlaxoSmithKline), a histamine receptor antagonist that also suppresses vascular expres sion of Eselectin, markedly and specifically improved survival of highrisk patients identified by tumour expression of sle a and sle x (REF. 45 ), further supporting the usefulness of selectins as therapeutic targets for cancer. Selectins and their ligands have also been reported to play key parts in the dissemination of haematological cancers 46 and the homing of leukaemic stem cells to microdomains within the bone marrow 47 . Eselectin is constitutively expressed in the bone marrow 48 and binds carbohydrate ligands that are found on leukaemic stem cells. Once adherent to these microdomains in the bone marrow, leukaemic cells become quiescent and less sus ceptible to killing by antiproliferative chemotherapy drugs such as cytosine arabinoside 49 . Potent selectin antagonists present new therapeutic opportunities for treating these diseases. By preventing sequestration of leukaemic cells in the bone marrow and keeping Glycomimetic A molecular mimic of a functional carbohydrate that has improved affinity for its target and drug-like pharmacokinetic properties. A large, synthetically produced polymer in which the atoms are arranged in many branches and subbranches radiating out from a central core. An effect that is exerted on a protein by the binding of an effector molecule at a site other than the protein's active site or binding site. them in circulation, combination therapy with selectin antagonists is likely to make the cells more susceptible to chemotherapy. Some examples of glycomimetic, small molecule antagonists of the selectins are presented in Mucosal surfaces present barriers to the environment that are potentially susceptible to infec tion. Migrating dendritic cells guard mucosal surfaces, capturing microorganisms and presenting processed antigens to activated T cells, thereby inducing an immune response against the invading pathogens. By screening a library of dendritic cellspecific mono clonal antibodies that inhibit binding to intercellular adhesion molecule 3 (ICAM3; an adhesion molecule that activates T cells), a single cell surface protein was discovered: DCSIGn 50 . The aminoacid sequence of DCSIGn is identical to a previously described HIv glycoprotein 120 (gp120) binding Ctype lectin 51, 52 . DCSIGn that is expressed on patrolling dendritic cells in the mucosa binds to carbohydrate structures on the gp120 protein coat of HIv, which is the initial entry port of HIv to the host. HIv particles bound to DCSIGn on the surface of den dritic cells are protected from destruction in the blood and migrate to the lymph nodes where they transinfect T cells through the CD4-CCR5 (CCchemokine recep tor 5) complex on the T cell surface 51 . The binding spe cificity of DCSIGn is for fucose and mannose residues, with higher affinity and specificity for the fucose linkage in le a/x type oligosaccharide structures. Formation of the active structure and binding of DCSIGn occurs in a Ca 2+ dependent manner 52, 53 . In addition to HIv, various other pathogens -such as the hepatitis C virus 54 , Dengue virus 55 , Ebola virus 56 , Marburg virus 57 , coronavirus (which causes severe acute respiratory syndrome) 58 and cytomegalovirus 59 , as well as bacteria such as Mycobacterium tuberculosis 60 and Helicobacter pylori 52 and yeast (Candida albicans)exploit DCSIGn to infect their host. More recently, even parasites such as Leishmania spp. 61 and Schistosoma mansoni 62 have also been shown to bind DCSIGn. The fact that different pathogens have capitalized on this infection strategy makes DCSIGn an interesting target for therapeutic intervention. In a study on the binding and transfer of HIv in human rectal mucosa cells, more than 90% of bound virus was bound to cells expressing DCSIGn, although these cells represented only 1-5% of the total mucosal mononuclear cells. Furthermore, DCSIGnspecific antibodies blocked more than 90% of HIv binding 63 . Other studies have shown that multivalent glycoconjugates of lewis x or dmannose prevented the attachment of Ebola or herpes virus to dendritic cells through DCSIGn and thus prevented the subsequent infection of immune cells [64] [65] [66] . Glycomimetic compounds that inhibit DCSIGn are based on two lead structures. The first are highmannose oligosaccharides and the second is lfucose as part of a lewis epitope 67 . These determinants are synthesized by pathogens to camouflage their appearance as host tissue. To improve the affinity and pharmacokinetic properties of these naturally occuring antagonists, glycomimetics of both types of ligands have been synthesized. Highdensity arrays of unbranched Manα(12)Man terminated oligosaccharides bind to DCSIGn almost as effectively as the entire Man 9 oligosaccharide (REF. 68 ). Therefore, the nonreducing end Manα(12)Man frag ment of Man 9 was suggested to play a crucial part in DCSIGn recognition. To mimic 1,2mannobiose, one hexose moiety was replaced by a cyclohexanediol derivative, leading to the pseudo1,2mannobioside compound 1 (FIG. 2) , which had a threefold greater affinity for DCSIGn than did 1,2mannobiose (half maximal inhibitory concentration (IC 50 ) = 0.62 mM and 1.91 mM, respectively) 69 . Furthermore, in infection studies using an in vivo model of Ebola infection, the glycomimetic compound 1 inhibited infection of DCSIGnexpressing Jurkat cells more efficiently than the corresponding natural disaccharide. Although the inhibitory concentration in these experiments was in the millimolar range, compound 1 might be useful in the preparation of highaffinity multivalent antagonists. Such an approach is encouraged by the strong inhibitory effects of multivalent antagonists on DCSIGn bind ing, as observed for dendritic mannose conjugates 70 or oligolysinebased oligosaccharide clusters 71 . Similarly, αfucosylamine linked to 2amino cyclo hexane carboxylic acid (compound 2) mimics lewis x trisaccharide and inhibits DCSIGn with a twofold greater potency (IC 50 = 0.35 mM and 0.8 mM, respec tively) 72 . These binding affinities are too weak for these compounds to have any therapeutic promise; however, when the oligosaccharides are displayed on large multi valent dendrimers, activity is greatly improved and bio logical activity can be shown in vitro 71 . Although such large multivalent presentations of carbohydrates or mimics thereof are a relatively simple means to increase activity, they pose a pharmaceutical challenge in terms of routes of administration and possible side effects, such as unwanted immune responses. A classical approach to discovering DCSIGn antago nists was successfully demonstrated by screening large libraries of small molecules in an automated assay format. By screening over 35,000 compounds, 7 hits with IC 50 values in the low micromolar range were identified, such as compound 3 and compound 4 (REF. 73 ). Interestingly, the structures of these hits bear no resemblance to the native carbohydrate ligands of oligomannose or the lewis epitopes and do not con tain functional groups to interact with Ca 2+ in the CRD. Their inhibitory activity could be caused by binding to other domains on DCSIGn, leading to an allosteric effect. Itype lectins are a family of carbohydratebinding proteins in the immunoglobulin superfamily, and include Siglecs 74 . The Siglecs function as cell signalling coreceptors and are primarily expressed on leuko cytes that mediate acquired and innate immune functions. The cytoplasmic domains of most Siglecs contain immunoreceptor tyrosinebased inhibitory motifs, which are characteristic of accessory proteins that regulate transmembrane signalling and endocytosis of cell surface receptor proteins. The diverse specificity for their sialoside ligands and variable cytoplasmic regulatory elements enable Siglecs to carry out unique roles at the cell surface. Siglecs can be broadly divided into an evolutionarily conserved group (Siglec 1 (also known as sialoadhesin), Siglec 2 (also known as CD22) and Siglec 4 (also known as MAG)) and a Siglec 3related A dynamic extension at the tips of axons that is supported by actin and grows towards synaptic targets. . The evolutionarily conserved group shows selective binding properties: Siglec 1 and MAG preferentially bind α(23)linked Nacetylneuraminic acid (neu5Ac) and Siglec 2 is highly specific for α(26)linked neu5Ac. By contrast, members of the Siglec 3related group are more promiscuous in their binding, often recognizing more than one presentation of neu5Ac. The most comprehensively characterized Siglecs are Siglec 2, a regulatory protein that prevents the over activation of the immune system and the development of autoimmune diseases, and MAG, a protein that blocks regeneration of the central nervous system (CnS) after injury 75 . Unlike the peripheral nervous system (PnS), the injured adult CnS inherently lacks the capacity for axon regeneration. Although neurite outgrowth is possible in principal, it is blocked by inhibitor proteins expressed on residual myelin and on astrocytes that are recruited to the site of injury. To date, three major inhibitor proteins have been identified: reticulon 4 (RTn4; also known as nogo A) 76 , myelin oligodendrocyte glycoprotein (MOG) 77 and MAG 78 . These three proteins bind to and activate the RTn4 receptor, which is located on the surface of the neuron. This leads to the formation of a complex with the nerve growth factor receptor (nGFR; also known as p75nTR) and the activation of the RhoA-ROCK (Rho associated, coiled coilcontaining protein kinase) cascade, which results in growth cone collapse 79 . The RhoA-ROCK inhibitory cascade can also be triggered by a complex formed by MAG, brain ganglio sides (especially GM1b, GD1a, GT1b, GT1β and GQ1bα) 80 and nGFR 81 . Although the exact biological role of the MAG-ganglioside interaction has yet to be resolved, in some systems inhibition of axon regenera tion by MAG could be completely reversed by sialidase treatment, suggesting that sialidated glycans are the main axonal ligands of MAG 82 . SAR studies [83] [84] [85] have revealed that the terminal tetrasaccharide epitope neu5Acα(2→3)Galβ(1→3)[neu5Acα(2→6)]GalNAc of GQ1bα shows superior binding to MAG compared with the terminal trisaccharide epitope, which is present in GD1a and GT1b, for example 86 . Further refinements of the SAR profile have led to the identification of MAG antagonists that have improved affinities and, at least in some cases, remarkably simple structures (FIG. 3) . However, owing to the use of different assay formats, it has been difficult to compare the reported affinities of these compounds for various ligands. Overall, starting from the lowaffinity tetrasaccharide lead structure compound 6 (REF. 87 Bacterial and viral lectins For colonization and subsequent development of an infectious disease, enteric, oral and respiratory bacteria require adhesion to the host's tissue. This grants them a substantially greater resistance to clearance and killing by immune factors, bacteriolytic enzymes and anti biotics. In addition, such bacteria are better able to acquire nutrients, further enhancing their ability to survive and infect the host. Therefore, antiadhesive drugs that prevent the adhe sion of pathogens to host tissues may offer a novel strat egy to fight infectious diseases 89 . The alarming increase in drugresistant bacterial pathogens makes a search for new approaches to fight bacterial infections essential 90 . . The inhibitory signal is transduced into the cytosol of the neuron through the co-receptor NGFR (nerve growth factor receptor; also known as p75NTR). MAG bound to the brain gangliosides GD1a, GT1b and GQ1bα also transduces the inhibitory signal, with the help of NGFR as a co-receptor, into the cytosol 79 . b | GQ1bα is the brain ganglioside with the highest affinity for MAG 80 ; replacement of its inner sialic acids by sulphates (to produce compound 5) led to a fourfold increase in affinity 165 . The tetrasaccharide compound 6 (REF. 87 ) is the minimal carbohydrate epitope of GQ1bα for MAG binding and has served as a lead structure for the development of antagonists; with compound 7, an excellent correlation between the degree of neurite outgrowth and the binding affinities was established 88 A type of decision-making process used by bacteria to coordinate gene expression and behaviour according to the local density of their population. A quantitative measurement of the disorder in a system. The change in heat divided by the absolute temperature is the entropy change or cost of the thermodynamic process. The non-sugar component that remains after hydrolysis of a glycoside. Because antiadhesive agents are not bacteri cidal, they are less likely to promote the propagation of resistant strains than bactericidal agents, such as antibiotics. The carbohydrate epitopes on the surface of host cells that are used by bacteria and viruses for colonization and infection (TABLE 2) are the starting point of the search for glycomimetic entry inhibitors. A challenge of antiadhesion therapy is that most pathogens possess genes encoding several types of adhesins, so that, during the infection process, they may express more than one of these adhesins. Glycomimetic antagonists that are designed to inhibit multiple adhesins are feasible to develop, and examples are described below for Pseudomonas aeruginosa. P. aeruginosa can be part of the normal flora in healthy adults but becomes a deadly pathogen in individuals who are immunocompromised, patients with cystic fibrosis and hospitalized, critically ill patients. An increasing percentage of P. aeruginosa infections are antibiotic resistant. For its adhesion to host cells, the pathogen expresses lectins such as PAIl and PAIIl 91 . These lectins are virulence factors under quorum sensing control and are, by themselves, cytotoxic to primary epithelial cells in culture 92 . At low concentrations, they inhibit ciliary beating of epithelial cells in explants of nasal polyps 93 . Inhibition can be completely reversed by treatment with the carbo hydrate ligand of the lectin. Thus, 24 hours after addition of fucose, ciliary beating returns to normal frequency 94 . PAIl and PAIIl are tetrameric lectins that require Ca 2+ for carbohydrate binding. The crystal structures of both lectins complexed with their carbohydrate ligands have been resolved (FIG. 4) . PAIl preferentially binds to terminal αlinked dgalactose in the presence of one Ca 2+ ion, whereas PAIIl binds with an unusually strong micromolar affinity to lfucose and requires two Ca 2+ ions 95, 96 . PAIl and PAIIl are soluble intracellu lar lectins. However, once released from the cells, these lectins cause bacteria to adhere to host tissue -a process that can be reversed by incubation with dgalactose and dmannose, respectively 97 . The native carbohydrate inhibitors of PAIl and PAIIl, dgalactose and lfucose, were successfully used to treat a tobramycinresistant P. aeruginosa infection in a case report 98 . Combination therapy of tobramycin with dgalactose and lfucose to inhibit the virulence factors PAIl and PAIIl cured an 18monthold infant with systemic and pulmonary infections, as determined by microbiological testing. Screening with the glycan arrays of the Consortium for Functional Glycomics revealed that the lewis a trisaccharide, Galβ (13) [Fucα (14) ]GlcNAc, is a high affinity ligand for PAIIl 99 , with a dissocation constant of 210 nM 100 . To reduce the complexity of the trisaccha ride antagonists, glycomimetics based on the Fucα(14) GlcNAc disaccharide -for example, the antagonist com pound 12 -were synthesized. By titration calorimetry experiments, increased entropy costs upon binding were detected as a result of the higher flexibility of Fucα (14) GlcNAc compared with lewis a . However, additional enthalpic interactions that originate from a network of hydrogen bonds compensate for this entropic penalty 101 . A further simplification of the PAIIl antagonists was achieved when αlfucosides bearing heterocyclic sub stituents as aglycons were synthesized. Surprisingly, some candidates -for example, compound 13 -have a similar potency to lewis a (REF. 99 ). Oligovalent forms of the Fucα (14)GlcNAc epitope, such as compound 14 (REF. 102 ), exhibit increased activity compared with monovalent forms; however, in most cases, this effect was only modest on a per saccharide basis. To date, multivalency has only been explored with dendri mers that present lfucose, which show an increase in affinity of up to a factor of 20 on a per saccha ride basis 103 . Finally, to prevent adhesion of P. aeruginosa mediated simultaneously by the PAIl and PAIIl lectins, hetero bifunctional ligands that present both dgalactose and lfucose in an oligovalent array (as in compound 15 (REF. 104 )) or as a smallmolecule glycomimetic (as in compound 16 (REF. 105 )) have been constructed. In a study to determine the efficacy of compound 16 in mice surgically stressed by 30% hepa tectomy, 60% of the control group died 48 hours after acute infection with P. aeruginosa, whereas 100% of mice treated with compound 16 survived 105 . FimH. Urinary tract infections (UTIs) are among the most prevalent inflammatory diseases that are caused by pathogens 106, 107 . The predominant pathogen in UTIs is uropathogenic Escherichia coli (UPEC), which causes more than 80% of all infections in otherwise healthy people (uncomplicated UTI). In healthy individuals, most uropathogens originate from the rectal microbiota and enter the normally sterile urinary bladder through the urethra, where they trigger the infection (cystitis). Once in the urinary tract, bacteria attach to the urinary tract epithelium through fimbrial adhesion molecules to avoid the host's defence mechanisms. Once bound, the bacteria are presumably internalized in an active process that is similar to phagocytosis 108 . Uncomplicated UTI can be effectively treated with oral antibiotics such as fluoroquinolones, cotrimoxazol or amoxicillin and clavanulate, depending on the sus ceptibility of the pathogen involved. However, recurrent infections and subsequent antibiotic exposure can result in the emergence of antimicrobial resistance, which often leads to treatment failure and reduces the range of therapeutic options. So, there is an urgent need for effi cient, costeffective and safe nonantibiotic therapy to prevent and treat UTIs without facilitating antimicrobial resistance. Inhibition of type 1 fimbriaemediated bacte rial attachment to the bladder epithelium is a promising approach to achieve this goal 109 . Studies showed that αmannosides are the primary bladder cell ligands for UPEC and that the attachment event requires the highly conserved FimH lectins, which are located at the tip of the bacterial fimbriae. A structure-function analysis showed that the residues of the FimH mannose binding pocket are invariant across 200 UPEC strains 110 . A non-covalent interaction between organic compounds that contain aromatic moieties. More than two decades ago, various oligomanno sides 111 and aromatic αmannosides 112 that antagonize type 1 fimbriaemediated bacterial adhesion were identified. Two approaches have been taken to improve their affinity: the rational design of ligands guided by information obtained from the crystal structure of FimH, and the multivalent presentation of the αmannoside epitope. The crystal structure of the FimH receptorbinding domain was solved in 1999 (REF. 113) and the corre sponding complex with oligomannoside3 (REF. 114) has recently become available. Despite this detailed know ledge of the binding event, few attempts to translate this information into lowmolecularmass antagonists have been reported 112, [115] [116] [117] . A selection of monovalent FimH antagonists is depicted in FIG. 5. The reference compound, methyl αdmannoside (compound 17) binds in the millimolar range 118 , but the most potent monovalent antagonist reported so far, compound 22, binds with nanomolar affinity 117 . The reported affinities can be explained on the basis of the structure of the CRD that is located on the tip of the FimH protein (FIG. 5) . First, the hydroxyl groups at the 2, 3, 4 and 6 positions of mannose form an extended hydrogen bond network 114, 118 . Second, the entrance to the binding site formed by two tyrosines and one isoleu cine -the socalled 'tyrosine gate' -supports hydro phobic contacts 118 . The aromatic aglycons of antagonists -as occur in compounds 20 and 21, for example -can establish energetically favourable π-π interactions with this tyrosine gate, leading to substantially improved affinities. A further enhancement of affinity was achieved using oligovalent and multivalent FimH antagonists (for example, compounds 23 to 28). Soluble FimH antagonists that are applied to prevent bacterial adhesion to the host tissue are faced with the challenge of mechanical forces resulting from fluid flow. It is commonly presumed that the duration of receptorligand interactions is shortened by shear stress. However, it was recently discovered that the ability of E. coli to avoid detachment is dramatically increased by shear stress 119 . As a consequence of shear stressenhanced adhesion, E. coli evades detachment from body surfaces by soluble glycoproteins or peptides that are ubiquitous in body fluids. An example is the glycoprotein uromodulin (also known as the Tamm-Horsfall urinary glycopro tein), which binds to FimH and is thought to function as a body defence against E. coli infections 120 . On the basis of simulations 121 , it is thought that forceinduced separation of FimH from its mannose ligand causes a conformational change of the binding pocket from a lowaffinity to a highaffinity conformation. Instead of the application of competitive antagonists, allosteric antagonists that are capable of stabilizing the lowaffinity conformation might lead to a successful therapy. Although monovalent and oligovalent antagonists with nanomolar affinity have been reported, there are no data available regarding their pharmacokinetic prop erties. However, for the treatment of UTI, oral bioavail ability and fast renal excretion to reach the targets in the urinary tract are prerequisites for therapeutic success. As in other fields that have spawned successful new therapeutics (for example, monoclonal antibodies), years of effort have been required to understand the unique challenges that are inherently linked to carbohydrate derived drugs and to develop the basic skills and the specific knowledge to move from the excitement of scientific discovery to the development of a new class of therapeutics. Although animal lectins usually show a high degree of specificity for glycan structures, their singlesite binding affinities are typically low. In biological systems, functional affinity is often attained by the oligovalent presentation of CRDs, either in an oligomeric protein (for example, cholera toxin 122 ) or through clustering at cell surfaces (for example, asialoglycoprotein recep tor 123 ). Additionally, the pharmacokinetic properties of carbohydrate hits, such as bioavailability or plasma halflife, are typically unsatisfactory for therapeutic applications. Finally, although tremendously improved novel glycosylation protocols 124 and solidphase approaches 125 have become available, oligosaccharides are still only manufactured by cumbersome multistep syntheses. Therefore, the challenge is to mimic the structural information of a functional carbohydrate with a com pound that has druglike characteristics. The first step in this process is to understand the SAR of a carbohydrate lead, specifically the contribution made by each func tional group to binding as well as the threedimensional presentation of the pharmacophores. Based on this information, it is possible to identify glycomimetics that are preorganized in their bioactive conformation -that is, which will adopt their bound conformation in solution. In addition, the mimics should show improved pharmacokinetic properties -in particular, improved bioavailability and serum halflife -while minimizing toxicity and cost of synthesis. In the past, the develop ment of carbohydratederived drugs was often not entirely focused on simultaneously solving all of the . As the RIPs were obtained from different assays (yeast agglutination, adherence to cell lines derived from human urinary bladder epithelium or guinea pig epithelial cells as well as surface plasmon resonance experiments with immobilized FimH), they should be compared with caution. The cross-relaxation between two nuclei, which is observable through the longitudinal magnetization of a given nucleus after a second nucleus is perturbed from equilibrium. (Saturation transfer difference nuclear magnetic resonance spectroscopy). An experiment in which spin-diffusion of the nuclear Overhauser effect spreads magnetization throughout the hydrogen nuclei of a receptor, which is partially transferred to the hydrogen nuclei of a binding ligand. above requirements and some highprofile failures resulted, notably in the field of selectin antagonists. nevertheless, rationally designed glycomimetics have the potential to reap the rewards of a relatively untapped source of novel therapeutics for wideranging and important biological and medical applications. Understanding native interactions. The starting point for the rational design of glycomimetics is the analysis of the binding characteristics of the carbohydrate-CRD binary complex. The threedimensional structure of the lectin or the carbohydrate-lectin complex has been solved for a number of therapeutically interesting targets. Thus, E, P and lselectin cocrystallized with sle x or PSGl1 (Pselectin glycoprotein ligand 1) 126 , siaload hesin cocrystallized with 3′sialyl lactose 127 , or DCSIGn cocrystallized with the pentasaccharide GlcNAc 2 Man 3 (REF. 128) hold valuable information for the rational design of glycomimetics. In cases in which the structure has not yet been solved, homology models can be generated -as is the case for MAG, for example 129 . Detailed insight into the binding event can be gained by nuclear magnetic resonance (nMR) experiments. For example, the bound conformation of a functional carbo hydrate ligand in the CRD of the target lectin can be deter mined using transferred nuclear Overhauser effect (nOE) 130 . In addition, the binding epitope can be identified by satu ration transfer difference nMR spectroscopy (STD NMR spectroscopy) 131 . This technique has been used to study interactions of carbohydrate ligands with the rotavirus receptor, vP8 (REF. 132 ), the anticarbohydrate tumour associated antibody GSlA1 (REF. 133 ), Eselectin 134 and MAG 87, 135 . Overall, transfer nOE nMR and STD nMR experiments allow a rapid insight into the binding char acteristics of carbohydrate-lectin interactions and can replace, at least partially, Xray investigations and the timeconsuming mapping of binding epitopes by chemical means 136 . Enhancing binding affinity. The generally low affinity of carbohydrate-lectin interactions is a consequence of shallow binding sites of lectins, leading to a high solvent accessibility of the complex forming hydrogen bonds and salt bridges. Owing to large offrates (k off ), the binary complexes are characterized by short dissociative halflives (t 1/2 ), typically in the range of seconds -as shown for selectins and their physiological ligands [137] [138] [139] , the carbo hydraterecognizing antibody GSlA1, sle a (REF. 133) and MAG antagonists 135 . Given that, for a thera peutic application, the t 1/2 of a drug-target binary com plex is expected to be in the range of minutes to a few hours, improving the k off of glycomimetic compounds is mandatory for therapeutic applications 140 . Often, mammalian lectins undergo numerous directed, but weak, interactions with their ligands. A specific example, the interaction of sle x with Eselectin, is outlined in FIG. 6a. It consists of six solventexposed hydrogen bridges and a salt bridge (to produce complex 29). One possible approach to improve affinity is to pre organize the antagonist in its bioactive conformation to compensate for the low enthalpic contributions by reducing the entropy costs on binding. For Eselectin, this strategy was successful (see complex 30 in FIG. 6a) . As elucidated by Xray 126 or STD nMR 134 studies, the GlcNAc moiety does not interact with the binding site and serves solely as a linker that positions the galactose and the fucose moiety in the correct spatial orientation. It was successfully replaced by noncarbohydrate link ers 141, 142 . In addition, steric repulsion deriving from properly placed substituents on the linker moiety can further improve the preorganization of the core and, as a result, the affinity of the corresponding antagonist 130 . Furthermore, the preorganization of the carboxylate was optimized as well, revealing (S)cyclohexyl lactic acid as the best mimic of neu5Ac 141 . If the target lectin offers a wellstructured binding pocket, the free energy of binding can be improved by incorporating additional enthalpic contributions. Successful examples are the neuraminidase inhibitors zanamivir 7 and oseltamivir 9 . For the influenza viral coat protein neuraminidase, the natural substrate neu5Ac and the corresponding glycal neu5Ac2en (compound 31), which mimics the transition state of the hydrolytic reaction, have only millimolar to micromolar affinities. The improved affinities of the transition state analogues zanamivir and oseltamivir result from a guanidinium substitution in the 4 position, enabling the forma tion of a new salt bridge 7 , or from the replacement of the glycerol side chain in the 6 position, leading to a new, favourable lipophilic interaction by induced fit 9 (FIG. 6b) . Finally, multivalency frequently occurs in nature and leads to tight binding in situations in which univa lent protein-ligand binding is weak [143] [144] [145] . Recognition of carbohydrate ligands by bacterial and mammalian lectins are examples of this phenomenon. For the spe cific inhibition of these recognition events, oligovalent ligands have been proposed (see, for example, FIGS 4, 5) . However, the design of tightbinding oligovalent ligands is, for the most part, an empirical endeavour. Tailored oligovalency, whereby the spacing of a limited number of tethered branches is matched to that between adja cent sugar binding sites of a protein or a protein cluster, potentially offers substantial increases in avidity for the target 143, 146, 147 . Pharmacokinetics. Unfortunately, only limited phar macokinetic data are reported for any carbohydrate or glycomimetic. For oral absorption by passive permeation through the membrane barrier of the small intestine 148 , there are limitations regarding molecular mass, polarity and the number of hydrogen bridge donors and accep tors 149 . The hydrophilic nature of oligosaccharides caused by the large number of hydroxyl groups and charges (sulphates and carboxylates) makes their oral availability virtually impossible. Therefore, when glyco mimetics are designed, the pharmacokinetic as well as the pharmacodynamic profile should be adjusted. Possible strategies to improve passive absorption are the bioisoteric replacement of crucial groups 150 or a pro drug approach 151 . A successful example of the prodrug approach is oseltamivir, which is an ester prodrug. Once Its absolute bioavailablity from the orally adminis tered prodrug is 80%. It is detectable in plasma within 30 minutes and reaches maximal concentrations after 3-4 hours 153 . In addition, the feasibility of using an activetransport system that is abundant in the intestine, liver, kidney or brain should also be considered 154 . Many drugs that are rationally designed or derived from natural products that cannot be absorbed by passive transport (such as βlactam antibiotics, heart glycosides or fungicides) take Figure 6 | enhancing the affinity of carbohydrate-derived drugs. a | The affinity of carbohydrate-derived drugs can be improved by pre-organization in the bioactive conformation. In solution, the core conformation (shown in red) of sialyl Lewis x is in the range of +10° to -60° and the acid orientation (shown in blue) is in the range of +80° to +150°. In the bioactive conformation (complex 29), the core conformation is approximately -40° and an acid orientation is approximately 110° . The degree of pre-organization of a mimetic in the bioactive conformation, as shown in complex 30, can be correlated with its affinity 130, 141 . b | Affinity can be improved by establishing new enthalpic interactions; comparisons of the binding mode of Neu5Ac2en (compound 31), zanamivir (Relenza) 7 and oseltamivir (Tamiflu) 9 to neuraminidase are depicted. bb, backbone; sc, side chains. advantage of active transport. In addition, active transport can be enforced by rational design -for example, by incorporating an amino acid into the structure and thereby creating a substrate for active transport by pep tide transporter 1 (PEPT1; also known as SlC15A1) and PEPT2 (also known as SlC15A2). A successful example is valacyclovir (valtrex/Zelitrex; GlaxoSmithKline), an antiviral drug used in the management of herpes sim plex, in which valine was attached to the parent drug acyclovir (Zovirax; GlaxoSmithKline/Biovail), leading to a fivefold increase of the oral availability 155 . Extensive analysis of the structural requirements of the PEPT1 transporter identified numerous analogues with higher affinity than valine; this information will be valuable for improving the oral availability of glycomimetics 156 . The usually short serum halflife and rapid excretion of carbohydrates presents an additional challenge for the design of glycomimetic drugs. Degradation in the pres ence of serum or liver microsomes are routine assays of metabolic stability that must be incorporated early in the design process of glycomimetics 157 . Organic anion and cation transport systems located in the liver and kidney are responsible for active excretion from the circulation 158 . The organic anion transporter family (OAT1 to OAT5) recognizes anions (specifically, carboxyl groups) connected to hydrophobic ring struc tures. RO640802, the active metabolite that is formed from oseltamivir, is an example of a glycomimetic drug with a serum halflife that is diminished by recognition and removal by the OAT system 159 . When probenecid, a competitive inhibitor of OAT1, is administered in com bination with oseltamivir, the serum halflife of the active metabolite is extended 160 . This strategy has been suggested to extend the supply of the US government's stockpile of oseltamivir in case of a national emergency in response to a pandemic outbreak of influenza. Both interactions with probenecid and specific transporter assays should be examined early in the development of a glycomimetic containing charged groups to identify structural elements that may adversely affect serum halflife. Recent efforts to elucidate the complexity and functions of the human glycome by pooling resources and tech nologies among academic centres has led to a rapid influx of discoveries and the acknowledgement of a new source of structural information that is not apparent from the human genome. The efforts in drug discovery reviewed here show the challenges in medicinal chemistry that need to be met for the development of druglike glycomimetics. Past efforts in this field have highlighted the drawbacks of using native oligosaccharides as drugs. Typically, both their pharmacodynamic and pharmacokinetic properties are insufficient for a therapeutic application. In addition to the lack of affinity, they suffer from low tissue permeability, short serum halflife and poor stability. Glycomimetics are designed to correct these shortcomings. The detailed insight into carbohydrate-lectin interactions that is required is predominantly provided by recent progress in nMR spectroscopy and Xray crystallography. Thus, the identification of the bound conformation of a func tional carbohydrate by transferred nOE nMR allows the design of mimetics with pharmaco phores that are preorganized in their bioactive conformation, leading to reduced entropy costs upon binding. By incorporating additional binding sites, which frequently leads to hydro phobic contacts, a further enhancement of affinity can often be achieved. Finally, the knowledge of the binding epitope as obtained by STD nMR allows the identifica tion of negligible and replaceable functional groups. As a consequence, the design of glycomimetics that have improved absorption, distribution, metabolism and excretion can be accomplished. Currently, these principles for the rational design of glycomimetics are being implemented in both academic institutions and industrial laboratories. As successful examples of glycomimetic drugs emerge, the strategies developed for their design will pave the way to real ize the potential of this relatively untapped source of therapeutics. Endothelial glycocalyx: sweet shield of blood vessels Measuring endothelial glycocalyx dimensions in humans: a potential novel tool to monitor vascular vulnerability The synthetic pentasaccharide fondaparinux: first in the class of antithrombotic agents that selectively inhibit coagulation factor Xa AO-128), one of the most important α-glucosidase inhibitors Miglitol: assessment of its role in the treatment of patients with diabetes mellitus Chemistry and biochemistry of microbial α-glucosidase inhibitors Rational design of potent sialidase-based inhibitors of influenza virus replication Metabolic soft spot identification and compound optimization in early discovery phases using Metasite and LC-MS/MS validation Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity This paper describes the design, synthesis and in vitro evaluation of transition-state-based inhibitors of influenza neuraminidase Identification of lectins from genomic sequence data Order out of complexity -protein structures that interact with heparin Order out of chaos: assembly of ligand binding sites in heparan sulfate Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1. Homology with the mouse lymphocyte homing receptor and other human adhesion proteins Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation Targeting selectins and selectin ligands in inflammation and cancer A review of the current understanding of selectins, their functions and role as targets for novel therapies A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion A carbohydrate domain common to both sialyl Le a and sialyl Le x is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1 A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma The tumor markers, sialyl Le a and sialyl Le x bind ELAM-1 Characterization of sialosylated Lewis x as a new tumor-associated antigen Prognostic value of CA-19-9 levels in patients with inoperable adenocarcinoma of the pancreas treated with gemcitabine Expression of carbohydrate antigen sialyl Le a : a new functional prognostic factor in gastric cancer Up-regulation of the oligosaccharide sialyl Lewis x : a new prognostic parameter in metastatic prostate cancer This paper describes functional human neutrophil receptors for E-selectin that are sialylated glycosphingolipids Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1 and CD-44 In vitro and in vivo selectinblocking activities of sulfated lipids and sulfated sialyl compounds Characterization of human platelet GMP-140 as a heparin-binding protein P-selectin binds to bacterial lipopolysaccharide GMP-140 binding to neutrophils is inhibited by sulfated glycans Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44 Oversulfated chondroitin/ dermatan sulfates containing GlcAβ1/IdoAα1-3GalNAc(4,6-O-disulfate) interact with L-and P-selectin and chemokines Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin Therapeutic effect of sulphated hyaluronic acid, a potential selectin-blocking agent, on experimental progressive mesangial proliferative glomerulonephritis Synthesis of a set of di-and trisulfated galabioses Pitfalls in the synthesis and biological evaluation of sialyl Lewis x mimetics as potential selectin antagonists A cautionary publication on a common pitfall in the design of antagonists of P-and L-selectins Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm CD44 on LS174T colon carcinoma cells possesses E-selectin ligand activity This study showed that CD44, a cancer stem cell marker, has E-selectin-binding activity Importance of E-selectin (ELAM-1) and sialyl Lewis a in the adhesion of pancreatic carcinoma cells to activated endothelium Expression of sialyl Lewis x , sialyl Lewis a , E-cadherin and cathepsin-D in human breast cancer: immunohistochemical analysis in mammary carcinoma in situ, invasive carcinoma and their lymph node metastasis Circulating KL-6/MUC1 mucin carrying sialyl Lewis a oligosaccharide is an independent prognostic factor in patients with lung adenocarcinoma Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose Cimetidine increases survival of colorectal cancer patients with high levels of sialyl Lewis x and sialyl Lewis a epitope expression on tumor cells A minor E-selectin ligand, CD65, is critical for extravascular infiltration of acute myeloid leukemia cells This study showed that CD44 is required for the homing of leukaemic cells to protective microdomains in the bone marrow The bone marrow is akin to skin: HCELL and the biology of hematopoietic stem cell homing Chemotherapy-resistant human AML stem cells home to and engraft within the bonemarrow endosteal region Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses The first study to provide insight into transient adhesion of dendritic cells and T cells mediated by DC-SIGN interaction DC-SIGN, a dendritic cellspecific HIV-1-binding protein that enhances transinfection of T cells Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells Specificity of DC-SIGN for mannose-and fucose-containing glycans DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2 DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus Specific asparagine-linked glycosylation sites are critical for DC-SIGN and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in human DCs, is a receptor for Leishmania amastigotes The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x Binding and transfer of human immunodeficiency virus by DC-SIGN + cells in human rectal mucosa Lewis x component in human milk binds DC-SIGN and inhibits HIV-1 transfer to CD4 + T lymphocytes Mannosyl glycodendritic structure inhibits DC-SIGN-mediated ebola virus infections in cis and in trans DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages Structural basis for distinct ligandbinding and targeting properties of the receptors DC-SIGN and DC-SIGNR By screening an extensive glycan array, the distinct ligand-binding properties of DC-SIGN and DC-SIGNR are shown Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycandependent gp120/protein interactions 1,2-Mannobioside mimic: synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding Oligolysine-based oligosaccharide clusters: selective recognition and elective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin Synthesis of novel DC-SIGN ligands with an alpha-fucosylamide anchor This study describes examples of small-molecule glycomimetic antagonists of DC-SIGN based on fucosylamides Non-carbohydrate inhibitors of the lectin DC-SIGN Siglecs and their role in the immune system A Review on the potential role of Siglecs in triggering endocytosis and in pathogen recognition Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1 Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth A novel role for myelinassociated glycoprotein as an inhibitor of axonal regeneration Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS This paper reports the discovery that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho Sialidase enhances spinal axon outgrowth in vivo Synthetic studies on sialoglycoconjugates 66: first total synthesis of a cholinergic neuron-specific ganglioside GQ1bα Total synthesis of a cholinergic neuron-specific ganglioside GT1aα: A high affinity ligand for myelin-associated glycoprotein (MAG) A highly efficient total synthetic route to α-series gangliosides: GM1α, GD1α, and GT1α Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (α-series) gangliosides and novel sulfated Chol-1 analogs Binding epitope of gangliosides to their neuronal receptor, myelin-associated glycoprotein, from saturation transfer difference NMR Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro Anti-adhesion therapy of bacterial diseases: prospects and problems Antibiotic resistance -the problem intensifies The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are 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aeruginosa lung infection by carbohydrate antagonists of the P. aeruginosa lectins PA-IL and PA-IIL Glycomimetics and glycodendrimers as high affinity microbial antiadhesins Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa X-ray structures and thermodynamics of the interaction of PA-IIL from Pseudomonas aeruginosa with disaccharide derivatives Synthesis and binding properties of divalent and trivalent clusters of the Lewis a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL Combinatorial variation of branching length and multivalency in a large (390 625 member) glycopeptide dendrimer library: ligands for fucose-specific lectins Synthesis of glycodendrimers containing both fucoside and galactoside residues and their binding properties to PA-IL and PA-IIL lectins from Pseudomonas aeruginosa Glycomimetic inhibitors of the PA-IL lectin, PA-IIL lectin or both of the lectins from pseudomonas Acute uncomplicated urinary tract infection in women Pathogenesis of urinary tract infection: an update Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins Carbohydrates as future anti-adhesion drugs for infectious diseases Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection Interaction of mannose-containing oligosaccharides with the fimbrial lectin of Escherichia coli Aromatic alpha-glycosides of mannose are powerful inhibitors of the adherence of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli Intervening with urinary tract infections using anti-adhesives based on the crystal structure of the FimH-oligomannose-3 complex Anti-adhesive compounds to prevent and treat bacterial infections. Patent application PCT WO Glycomimetics and glycodendrimers as high affinity microbial antiadhesins Evaluation of the carbohydrate recognition domain of the bacterial adhesin FimH: design, synthesis and binding properties of mannoside ligands This study details an analysis of the binding site of FimH adhesin and the identification of antagonists that have potency in the low nanomolar range Elevated shear stress protects Escherichia coli cells adhering to surfaces via catch bonds from detachment by soluble inhibitors Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors Beyond induced-fit receptor-ligand interactions: structural changes that can significantly extend bond lifetimes Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin The asialoglycoprotein receptor: a model for endocytic transport receptors Carbohydrates in Chemistry and Biology: A Handbook in 4 Volumes Automated synthesis of oligosaccharides as a basis for drug discovery Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P-and E-selectin bound to SLe X and PSGL-1 Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 Å resolution Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR Consistent bioactive conformation of the Neu5Acα(2→3)Gal epitope upon lectin binding Preorganization of the bioactive conformation of sialyl Lewis x analogues correlates with their affinity to E-selectin Characterization of ligand binding by saturation transfer difference spectroscopy STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core Comparative epitope mapping with saturation transfer difference NMR of sialyl Lewis a compounds and derivatives bound to a monoclonal antibody Epitope mapping of sialyl Lewis X bound to E-selectin using saturation transfer difference NMR experiments Examination of the biological role of the α(2-6)-linked sialic acid in gangliosides binding to the myelin-associated glycoprotein A review of synthetic glycomimetics that bind the influenza neuraminidase, myelin-associated glycoprotein and selectins Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1 Affinity, kinetics and thermodynamics of E-selectin binding to E-selectin ligand-1 Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1 Drugtarget residence time and its implications for lead optimization Development of tools for the design of selectin antagonists Diglycosylated 1,2-diols as mimetics of sialyl Lewis x and sialy Lewis a . 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Novel selectin blocker as potential therapeutics for inflammatory disorders Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation The war against influenza: discovery and development of sialidase inhibitors Human parainfluenza viruses hPIV1 and hPIV3 bind oligosaccharides with α2-3 linked sialic acids that are distinct from those bound by H5 avian influenza virus hemagglutinin Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus Role of sialic acids in rotavirus infection Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type I NMR experiments reveal the molecular basis of receptor recognition by a calicvirus Sialic acids as receptor determinants for coronaviruses The murid herpesvirus-4 gH/gL binds to glycosaminoglycans Sialic acid is a cellular receptor for Coxsackievirus A24 variant, an emerging virus with pandemic potential Binding of human papilloma virus L1 virus-like particles to dendritic cells is mediated through heparan sulfates and induces immune activation Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus) Structural basis of GM1 ganglioside recognition by simian virus 40 Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus The authors declare competing financial interests: see web version for details.