DOI: 10.1002/cmdc.201500107
Viewpoints
Glycans in Medicinal Chemistry: An Underexploited Resource Alberto Fernndez-Tejada,*[a] F. Javier CaÇada,[a] and Jesffls Jim¦nez-Barbero*[b, c] The biological relevance of glycans as mediators of key physiological processes, including disease-related mechanisms, makes them attractive targets for a wide range of medical applications. Despite their important biological roles, especially as molecular recognition elements, carbohydrates have not been fully exploited as therapeutics mainly due to the scarcity of structure–activity correlations and their non-drug-like properties. A more detailed understanding of the complex carbohy-
drate structures and their associated functions should contribute to the development of new glycan-based pharmaceuticals. Recent significant progress in oligosaccharide synthesis and chemical glycobiology has renewed the interest of the medicinal chemistry community in carbohydrates. This promises to increase our possibilities to harness them in drug discovery efforts for the development of new and more effective, synthetic glycan-based therapeutics and vaccines.
Carbohydrates are ubiquitous in nature and represent the most abundant class of biomolecules found in living organisms. They can exist as free monosaccharides, oligosaccharides, polysaccharides, or as essential components of glycoconjugates, including glycoproteins, glycolipids and glycosylated natural products.[1] The biosynthesis of glycans is not under direct genetic control but rather is regulated by the action of a wide array of enzymes dictated by metabolism, signal transduction, and cellular status. As a result, carbohydrates and glycoconjugates are typically heterogeneous. Moreover, they incorporate a wide range of monosaccharide residues that can be connected by glycosidic linkages with multiple variations in stereochemistry, regiochemistry, and branching that further contributes to their significant structural diversity. This variety in structures enables glycans to encode information that is essential in a number of physiological processes, such as protein folding, cell signaling, cell proliferation and differentiation, and tissue development.[2] These processes critically depend on molecular recognition events involving specific receptor– ligand interactions in which carbohydrates are key partners. Glycan-mediated interactions are also at the heart of medicinally relevant processes including bacterial adhesion, viral infection, inflammation, and immune system activation. As a consequence, carbohydrates are recognized to play pivotal roles in human health, being implicated in the development and progression of chronic (e.g., diabetes), neurodegenerative (e.g., Alzheimer’s) and infectious (e.g., influenza, malaria) diseases, as
well as cancer and congenital genetic disorders.[3] However, despite the biological and medical relevance of glycans, their structural complexity and heterogeneity has hampered the understanding of the molecular basis governing their functions. The relative scarcity of biochemical and analytical tools to decipher the biology that is concealed within their structures is another important obstacle in this direction. For these reasons, the pace of development of glycan-based platforms for diagnostic and therapeutic applications has hitherto been relatively slow. In addition, the intrinsic unfavorable pharmacokinetic properties of carbohydrates have further delayed their exploitation in medicinal chemistry as a source of new drugs.[4] Fortunately, recent advances in the broad field of glycoscience, including novel approaches in carbohydrate chemistry (glycochemistry) and biology (glycobiology), have considerably increased our understanding of the complex correlation between glycan structure and function.[5] However, while improved methods in enzymatic and chemical synthesis of oligosaccharides have been developed over the past years that enabled access to a wide range of complex carbohydrate structures, more widely applicable synthetic strategies are needed to produce larger quantities of glycans for in-depth structure– activity relationships studies. Similarly, the development of new biochemical tools to probe modifications in cell-surface glycosylation related to cancer, inflammation, and infection is necessary to identify unique glycan markers of transformed cells that can be exploited for the discovery of novel diagnostic and therapeutic approaches.[6] Determining the basis of protein–carbohydrate interactions is also important to improve our understanding of glycanmediated molecular recognition processes. In recent years, carbohydrate microarrays have been central in this respect, enabling the rapid analysis of binding events that involve cell-surface oligosaccharides. Microarrays have been widely used for a number of purposes, including the characterization of the sugar binding specificities of many lectins, enzymes and antibodies, detection of specific cells and pathogens (e.g., influenza virus, Leishmania) interacting with glycans, and identifica-
[a] Dr. A. Fernndez-Tejada, Prof. Dr. F. J. CaÇada Chemical and Physical Biology Centro de Investigaciones Biolûgicas (CIB-CSIC) Ramiro de Maeztu 9, 28040 Madrid (Spain) E-mail: [email protected] [b] Prof. Dr. J. Jim¦nez-Barbero CIC bioGUNE: Center for Cooperative Research in Biosciences Bizkaia Technology Park, 48160 Derio (Spain) E-mail: [email protected] [c] Prof. Dr. J. Jim¦nez-Barbero Ikerbasque, Basque Foundation for Science Mara Lûpez de Haro 13, 48009 Bilbao (Spain)
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Viewpoints tion of anti-glycan antibodies associated with a range of diseases such as malaria and cancer. Therefore, it becomes apparent that these carbohydrate array technologies hold great promise for medical applications, not only as diagnostic tools to identify disease-related glycan biomarkers and to detect these markers in patient samples, but also as a useful platform to obtain valuable information for drug discovery purposes as well as for the development of oligosaccharide-based vaccines.[7] Despite the excellent role of glycans as recognition molecules, the exploitation of many pathophysiologically relevant sugar–protein interactions has not yet reached its full potential. Thus, carbohydrates represent a promising but relatively untapped source of new drug targets, as demonstrated by the fact that few carbohydrate-based pharmaceuticals are currently on the market.[8] Beyond the synthetic challenges associated with their structures, one of the main reasons is the suboptimal physicochemical properties of sugars as drug candidates, including their weak monovalent binding affinities, their hydrophilic nature and low tissue permeability, and their short serum half-lives. In addition, another barrier to the full exploitation of the broad potential of complex glycans for the development of effective pharmaceuticals involves overcoming the basic challenges associated with the elucidation of relevant, detailed structure–activity relationships in carbohydrates.[9] This is mainly due to their vast structural heterogeneity and functional complexity, along with the relative scarcity of chemical and analytical tools to manipulate and accurately measure these complex glycan structures. Most of these drawbacks have recently started to be addressed with the rational design of smaller-sized carbohydrate derivatives and mimics that have improved drug-like properties.[10]
The development of such glycomimetics requires extensive knowledge of the conformation and three-dimensional structure of glycans and, more importantly, of the molecular basis of the interaction with their protein receptors (i.e., antibodies, enzymes or lectins). Two major experimental techniques for this atomic-level characterization are NMR spectroscopy and Xray crystallography. The transferred NOE (tr-NOESY) method allows to determine the bound conformation of the sugar at the protein binding site, whereas the saturation transfer difference (STD) NMR gives information on the specific sites of the glycan (ligand epitope) that are in close contact with the receptor.[11] Information gained from these experiments is usually complemented with computational modeling, for example molecular dynamics simulations, to more accurately deduce the bioactive glycan conformation. This knowledge can thus be used for medicinal chemistry and drug discovery efforts in the design and synthesis of new glycomimetics with enhanced binding affinity or inhibitory properties and improved synthetic accessibility. Several successful examples of carbohydrate-based drugs include the influenza virus neuraminidase inhibitors zanamivir and oseltamivir, and the glucosidase inhibitors acarbose and miglitol against diabetes (Figure 1).[12] Another related iminosugar, miglustat, inhibits not only a-glucosidase but also ceramide glucosyltransferase, and is used for the treatment of lysosomal storage disorders (LSDs), namely Gaucher and NiemannPick C diseases.[13] These pathologies are associated with abnormal glycolipid metabolism due to defects in the activity/ trafficking of lysosomal hydrolases, which results in intracellular accumulation of glucosylceramide and glycosphingolipids, respectively. While replacement of the deficient enzyme (glucocerebrosidase in Gaucher patients) and the substrate reduction
Figure 1. Some monosaccharide and oligosaccharide mimics (“glycomimetics”) currently on the market as important examples of approved, small-molecule carbohydrate-based drugs.
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Viewpoints therapy (SRT) have proved to be effective in the treatment of some LSDs, synthetic monosaccharide mimics able to restore the activity of those pathologically mutated glycosidases are already under clinical evaluation and constitute the basis of the chemical chaperoning therapeutic approach.[14] In addition to these monosaccharide-derived glycomimetics, heparin is another blockbuster drug that has tremendously benefited from the power of medicinal chemistry. Structure–activity investigations and extensive understanding of heparin’s mode of action enabled access to a family of low-molecularweight heparins (LMWHs) with improved anticoagulant function. Synthetically defined and therapeutically active carbohydrate mimics have also been developed, by chemical and chemoenzymatic methods, that incorporate the precise heparin oligosaccharide sequence that is known to interact with antithrombin III as the basis of its anticoagulant activity.[15] The first example of such synthetic heparin analogues, the pentasaccharide fondaparinux, exhibited enhanced potency and better safety profile, and has been on the market since 2002 (Figure 1).[16] The heparin full polysaccharide has also been shown to have several other activities and roles in different diseases, such as cancer, infection, and inflammation, which reveals the potential for additional medicinal chemistry studies on the heparin scaffold for the discovery of new therapeutic approaches. Besides free carbohydrates, glycosylated proteins hold great promise for use as pharmaceutical agents. In particular, the modification of intracellular proteins at their serine or threonine residues with O-linked b-N-acetylglucosamine (O-GlcNAc), which is controlled by two enzymes [O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)] is attracting a great deal of interest in the medicinal community because of its altered regulation in Alzheimer’s disease, cancer, and diabetes.[17] Despite the development of some chemical approaches to study this modification, progress in understanding the functional roles of O-GlcNAc in the cell has been slow and more efficient tools are still required.[18] While small molecule regulation of these enzymes is still underdeveloped, the recent discovery by Vocadlo and co-workers of an OGA inhibitor that slows neurodegeneration by increasing O-GlcNAc levels in mouse brain has identified OGA as an important drug target in Alzheimer’s disease.[19] The field of therapeutic glycoproteins constitutes another relevant area for biomedical research and drug development. In these glycoconjugates, the carbohydrate moiety is essential to modulate the properties of the protein, including folding, solubility, stability and biological activity.[20] However, the heterogeneous nature of recombinantly derived glycoproteins complicates the understanding and elucidation of the influence of specific carbohydrates and their sites of glycosylation on the protein therapeutic action. In fact, different glycosylated variants (glycoforms) of a protein drug have distinct physicochemical and biological properties, and given their complexity, detailed structure–function relationships in this type of pharmaceuticals are lacking. As a consequence, recent efforts in the field have focused on the preparation and evaluation of homogeneous glycoproteins that can yield useful structure–activity ChemMedChem 2015, 10, 1291 – 1295
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correlations for advancing our knowledge on the impact of specific sugars on the protein biological function. Given the importance of protein glycosylation, several strategies have been pursued over the past years in an attempt to control this ubiquitous modification to produce tailor-made, fully active glycoprotein drugs. These approaches have been extensively reviewed elsewhere,[21] and include recombinant expression by genetic manipulation of the cell glycosylation machinery, in vivo metabolic oligosaccharide engineering for the incorporation of artificial monosaccharides into natural glycoproteins in the cell, in vitro glycan remodeling using exogenous enzymes, and synthetic chemistry methodologies based on chemoselective ligation protocols and de novo total synthesis. Because the efficacy of protein biopharmaceuticals critically depend on their specific glycoforms, some of the previous strategies have been applied for the production of improved therapeutic glycoproteins, being the blockbuster anti-anemia drug erythropoietin (EPO) and monoclonal antibodies some of the most representative cases. EPO is a 166-amino-acid-long glycoprotein containing four glycosylation sites (three N-linked and one O-linked) that has been widely used in recombinant form (epogen) for the stimulation of red blood cell formation. On the basis of previous studies revealing the key importance of glycosylation (or lack thereof) on EPO pharmacokinetics and in vivo stability, particularly the extent of sialylation on its N-linked sugars, a new version of the drug incorporating additional N-glycosylation sites (darbepoetin) was produced by genetic engineering that possessed improved therapeutic properties, namely prolonged serum half-life and increased potency.[22] To overcome the heterogeneity issues associated with biologically derived material, Danishefsky and co-workers recently accomplished the chemical synthesis of a fully glycosylated and active EPO in homogeneous, pure form.[23] Another example of the therapeutic potential of carbohydrates is their essential role in determining the effector functions and inflammatory activities of monoclonal antibodies. For instance, deglycosylation of immunoglobulin G has been shown to decrease binding to Fc (fragment crystallizable) protein receptors located on the immune cell surface, thereby abrogating antibody effector functions and decreasing inflammation, which has led to the investigation of therapeutic deglycosylated antibodies.[24] Sialylation of the Fc N-glycans also attenuates Fc receptor binding and induces anti-inflammatory effects, while defucosylation causes the opposite effect.[25] Thus, although these complex biomolecules may be viewed as outside of the medicinal chemistry field of action, the previous examples show that by understanding the effect of glycosylation on glycoprotein pharmaceuticals and through rational manipulation of their carbohydrate structures, it is possible to modify their biological functions and maximize their therapeutic potential. Considering the relevance of cell-surface carbohydrates as elements of molecular recognition by the immune system, glycans have also been key actors in this area for the development of oligosaccharide-based vaccines.[26] However, due to the poor immunogenicity of carbohydrates, which mainly elicit
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Viewpoints B-cell-dependent immune responses alone, their covalent attachment to protein carriers, and also co-administration with appropriate adjuvants, has enabled the generation of glycoconjugate vaccines that can elicit more potent, T-cell-mediated responses. Recent advances in carbohydrate chemistry together with the discovery of more efficient synthetic and biochemical tools has facilitated the design and synthesis of several vaccine candidates against infectious diseases produced by bacteria (Haemophilus influenza, Streptococcus pneumoniae, Mycobacterium tuberculosis), and parasites (Plasmodium falciparum, Leishmania).[27] Much research efforts have also been devoted to develop carbohydrate-based anti-HIV vaccines based on the discovery of potent broadly neutralizing antibodies (BnAb) against HIV-1, namely 2G12, and most recently PG9 and PG16. The increasing molecular understanding of the interaction between these BnAbs and the glycan structures on the viral surface has driven the design and synthesis of many oligosaccharide and glycopeptide antigens as epitope mimics for HIV-1 vaccine development.[28] While most of these synthetic derivatives showed good binding affinity for the relevant antibody, all attempts to elicit BnAbs by immunization have failed thus far. Nevertheless, these studies have yielded useful structure–activity relationships for carbohydrate antigenicity and also provided important insights into the glycan specificities for recognition by these antibodies.[29] With this valuable structural information and increasingly better tools in the glycoscience field, it is hoped that medicinal chemistry efforts in the characterization and reconstitution of minimal neutralizing epitopes by chemically defined glycan structures will provide new opportunities for HIV vaccine design. Carbohydrate-based anticancer vaccines have been the subject of intensive research for decades and have gained even greater interest over the last few years. This burgeoning field has been fueled by recent advances in glycobiology and glycochemistry that have contributed to identify an increasing number of tumor-associated carbohydrate antigens (TACAs), which are abnormally expressed in the surface of cancer cells but not in normal cells. Using the tools of chemical synthesis, a number of research groups around the globe have targeted these TACA structures to develop anticancer vaccine candidates.[30] Again, conjugation of the carbohydrate antigens to a protein carrier or another immunogenic delivery system, in combination with a potent immunoadjuvant (e.g., QS-21),[31] has been one of the most common approaches to produce robust T-cell-dependent immune responses. Several forms of TACAs have been explored, either as single or clustered entities, or even as multi-antigenic systems or unnatural, chemically modified variants. After a number of moderately successful two-component self-adjuvanting vaccines, where the B-cell epitope (Tn antigen) was linked to a Pam3Cys lipopeptide serving as an immunostimulant, fully synthetic three-component vaccines have recently been developed. These designs incorporate all the requisite elements necessary for strong immune activation, that is, B-cell and T-cell epitopes as well as a built-in adjuvant. As a significant example, the Boons tripartite vaccine was able to generate potent humoral and cellular responses ChemMedChem 2015, 10, 1291 – 1295
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and to decrease tumor size in mice, which gives some hope for future clinical trials in cancer patients.[32] Several other multicomponent constructs have also resulted in excellent immune responses,[33] which signals the promise of this fully synthetic strategies as potential carbohydrate-based vaccines for cancer immunotherapy. In conclusion, this Viewpoint article hopes to highlight the booming development of glycans at the interface of medicinal chemistry and chemical biology. The remarkable examples outlined herein are intended to illustrate the great promise of carbohydrates, both alone and as main constituents of other glycoconjugates, namely glycoproteins, as the next frontier in pharmaceutical discovery. The fields of glycobiology as well as carbohydrate and glycoprotein synthesis have made a comeback and have a bright future, with novel findings on the physiological roles of glycans and improved chemical and biological tools already appearing on the horizon.
Acknowledgments Generous financial support of the authors’ research is provided by the European Commission (Marie Curie International Outgoing Fellowship to A.F-T.), and the Spanish Ministry of Economy and Competitiveness (MINECO) (grant CTQ2012-32025). Keywords: carbohydrates · chemical glycobiology · drug discovery · glycans · medicinal chemistry · oligosaccharide synthesis [1] Glycoscience: Chemistry and Chemical Biology, 2nd ed. (Eds.: B. O. FraserReid, K. Tatsuta, J. Thiem, G. L. Cot¦, S. Flitsch, Y. Ito, H. Kondo, S.-i. Nishimura, B.Yu), Springer, Berlin, 2009. [2] Essentials of Glycobiology, 2nd ed. (Eds.: A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart, M. E. Etzler), Cold Spring Harbor Laboratory Press, New York, 2009. [3] D. Sols, N. V. Bovin, A. P. Davis, J. Jim¦nez-Barbero, A. Romero, R. Roy, K. Smetana, H.-J. Gabius, Biochim. Biophys. Acta 2015, 1850, 186 – 235. [4] L. Cipolla, F. Peri, Mini-Rev. Med. Chem. 2011, 11, 39 – 54. [5] J. E. Hudak, C. R. Bertozzi, Chem. Biol. 2014, 21, 16 – 37. [6] a) L. X. Wang, B. G. Davis, Chem. Sci. 2013, 4, 3381 – 3394; b) D. H. Dube, C. R. Bertozzi, Nat. Rev. Drug Discovery 2005, 4, 477 – 488. [7] a) S. Park, J. C. Gildersleeve, O. Blixt, I. Shin, Chem. Soc. Rev. 2013, 42, 4310 – 4326; b) C. D. Rillahan, J. C. Paulson, Annu. Rev. Biochem. 2011, 80, 797 – 823. [8] M. C. Galan, D. Benito-Alfonso, G. M. Watt, Org. Biomol. Chem. 2011, 9, 3598 – 3610. [9] Z. Shriver, S. Raguram, R. Sasisekharan, Nat. Rev. Drug Discovery 2004, 3, 863 – 873. [10] J. L. Magnani, B. Ernst, Discov. Med. 2009, 8, 247 – 252. [11] a) L. Unione, S. Galante, D. Diaz, F. J. CaÇada, J. Jim¦nez-Barbero, Med. Chem. Commun. 2014, 5, 1280 – 1289; b) V. Roldûs, F. J. CaÇada, J. Jim¦nez-Barbero, ChemBioChem 2011, 12, 990 – 1005. [12] B. Ernst, J. L. Magnani, Nat. Rev. Drug Discovery 2009, 8, 661 – 677. [13] a) T. D. Butters, R. A. Dwek, F. M. Platt, Glycobiology 2005, 15, 43R – 52R; b) T. D. Butters, R. A. Dwek, F. M. Platt, Curr. Top. Med. Chem. 2003, 3, 561 – 574. [14] a) Y. Suzuki, Brain Dev. 2013, 35, 515 – 523; b) L. Cipolla, A. C. Araffljo, D. Bini, L. Gabrielli, L. Russo, N. Shaikh, Expert Opin. Drug Discovery 2010, 5, 721 – 737. [15] R. J. Linhardt, J. Liu, Curr. Opin. Pharmacol. 2012, 12, 217 – 219. [16] M. Petitou, C. A. van Boeckel, Angew. Chem. Int. Ed. 2004, 43, 3118 – 3133; Angew. Chem. 2004, 116, 3180 – 3196.
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Viewpoints [17] G. W. Hart, C. Slawson, G. Ramirez-Correa, O. Lagerlof, Annu. Rev. Biochem. 2011, 80, 825 – 858. [18] a) P. S. Banerjee, G. W. Hart, J. W. Cho, Chem. Soc. Rev. 2013, 42, 4345 – 4357; b) F. Corzana, A. Fernndez-Tejada, J. H. Busto, G. Joshi, A. P. Davis, J. Jim¦nez-Barbero, A. Avenoza, J. M. Peregrina, ChemBioChem 2011, 12, 110 – 117. [19] a) S. A. Yuzwa, X. Shan, M. S. Macauley, T. Clark, Y. Skorobogatko, K. Vosseller, D. J. Vocadlo, Nat. Chem. Biol. 2012, 8, 393 – 399; b) S. A. Yuzwa, D. J. Vocadlo, Chem. Soc. Rev. 2014, 43, 6839 – 6858. [20] a) R. J. Sol, K. Griebenow, J. Pharm. Sci. 2009, 98, 1223 – 1245; b) R. Jefferis, Arch. Biochem. Biophys. 2012, 526, 159 – 166. [21] a) D. P. Gamblin, E. M. Scanlan, B. G. Davis, Chem. Rev. 2009, 109, 131 – 163; b) J. R. Rich, S. G. Withers, Nat. Chem. Biol. 2009, 5, 206 – 215. [22] S. Elliott, T. Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, L. Busse, D. Chang, J. Fuller, J. Grant, N. Hernday, M. Hokum, S. Hu, A. Knudten, N. Levin, R. Komorowski, F. Martin, R. Navarro, T. Osslund, G. Rogers, N. Rogers, G. Trail, J. Egrie, Nat. Biotechnol. 2003, 21, 414 – 421. [23] P. Wang, S. W. Dong, J.-H. Shieh, E. Peguero, R. Hendrickson, M. A. S. Moore, S. J. Danishefsky, Science 2013, 342, 1357 – 1360. [24] M. Crispin, Proc. Natl. Acad. Sci. USA 2013, 110, 10059 – 10060. [25] a) X. R. Jiang, A. Song, S. Bergelson, T. Arroll, B. Parekh, K. May, S. Chung, R. Strouse, A. Mire-Sluis, M. Schenerman, Nat. Rev. Drug Discovery 2011, 10, 101 – 111; b) R. Jefferis, Nat. Rev. Drug Discovery 2009, 8, 226 – 234. [26] M. L. Hecht, P. Stallforth, D. V. Silva, A. Adibekian, P. H. Seeberger, Curr. Opin. Chem. Biol. 2009, 13, 354 – 359.
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[27] a) P. Stallforth, B. Lepenies, A. Adibekian, P. H. Seeberger, J. Med. Chem. 2009, 52, 5561 – 5577; b) C. Anish, B. Schumann, C. L. Pereira, P. H. Seeberger, Chem. Biol. 2014, 21, 38 – 50. [28] a) A. Fernndez-Tejada, B. F. Haynes, S. J. Danishefsky, Exp. Rev. Vaccines 2015, DOI: 10.1586/14760584.2015.1027690; b) S. Horiya, I. S. MacPherson, I. J. Krauss, Nat. Chem. Biol. 2014, 10, 990 – 999. [29] a) B. Aussedat, Y. Vohra, P. K. Park, A. Fernndez-Tejada, S. M. Alam, S. M. Dennison, F. H. Jaeger, K. Anasti, S. Stewart, J. H. Blinn, H. X. Liao, J. G. Sodroski, B. F. Haynes, S. J. Danishefsky, J. Am. Chem. Soc. 2013, 135, 13113 – 13120; b) M. N. Amin, J. S. McLellan, W. Huang, J. Orwenyo, D. R. Burton, W. C. Koff, P. D. Kwong, L. X. Wang, Nat. Chem. Biol. 2013, 9, 521 – 526. [30] a) R. M. Wilson, S. J. Danishefsky, J. Am. Chem. Soc. 2013, 135, 14462 – 14472; b) T. Buskas, P. Thompson, G. J. Boons, Chem. Commun. 2009, 5335 – 5349; c) N. Gaidzik, U. Westerlind, H. Kunz, Chem. Soc. Rev. 2013, 42, 4421 – 4442. [31] a) G. Ragupathi, J. R. Gardner, P. O. Livingston, D. Y. Gin, Expert Rev. Vaccines 2011, 10, 463 – 470; b) A. Fernndez-Tejada, E. K. Chea, C. George, N. Pillarsetty, J. R. Gardner, P. O. Livingston, G. Ragupathi, J. S. Lewis, D. S. Tan, D. Y. Gin, Nat. Chem. 2014, 6, 635 – 643. [32] V. Lakshminarayanana, P. Thompson, M. A. Wolfert, T. Buskas, J. M. Bradley, L. B. Pathangey, C. S. Madsen, P. A. Cohen, S. J. Gendler, G. J. Boons, Proc. Natl. Acad. Sci. USA 2012, 109, 261 – 266. [33] Z. Yin, X. Huang, J. Carbohydr. Chem. 2012, 31, 143 – 186. Received: March 9, 2015 Published online on May 14, 2015
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Viewpoints
Glycans in Medicinal Chemistry: An Underexploited Resource Alberto Fernndez-Tejada,*[a] F. Javier CaÇada,[a] and Jesffls Jim¦nez-Barbero*[b, c] The biological relevance of glycans as mediators of key physiological processes, including disease-related mechanisms, makes them attractive targets for a wide range of medical applications. Despite their important biological roles, especially as molecular recognition elements, carbohydrates have not been fully exploited as therapeutics mainly due to the scarcity of structure–activity correlations and their non-drug-like properties. A more detailed understanding of the complex carbohy-
drate structures and their associated functions should contribute to the development of new glycan-based pharmaceuticals. Recent significant progress in oligosaccharide synthesis and chemical glycobiology has renewed the interest of the medicinal chemistry community in carbohydrates. This promises to increase our possibilities to harness them in drug discovery efforts for the development of new and more effective, synthetic glycan-based therapeutics and vaccines.
Carbohydrates are ubiquitous in nature and represent the most abundant class of biomolecules found in living organisms. They can exist as free monosaccharides, oligosaccharides, polysaccharides, or as essential components of glycoconjugates, including glycoproteins, glycolipids and glycosylated natural products.[1] The biosynthesis of glycans is not under direct genetic control but rather is regulated by the action of a wide array of enzymes dictated by metabolism, signal transduction, and cellular status. As a result, carbohydrates and glycoconjugates are typically heterogeneous. Moreover, they incorporate a wide range of monosaccharide residues that can be connected by glycosidic linkages with multiple variations in stereochemistry, regiochemistry, and branching that further contributes to their significant structural diversity. This variety in structures enables glycans to encode information that is essential in a number of physiological processes, such as protein folding, cell signaling, cell proliferation and differentiation, and tissue development.[2] These processes critically depend on molecular recognition events involving specific receptor– ligand interactions in which carbohydrates are key partners. Glycan-mediated interactions are also at the heart of medicinally relevant processes including bacterial adhesion, viral infection, inflammation, and immune system activation. As a consequence, carbohydrates are recognized to play pivotal roles in human health, being implicated in the development and progression of chronic (e.g., diabetes), neurodegenerative (e.g., Alzheimer’s) and infectious (e.g., influenza, malaria) diseases, as
well as cancer and congenital genetic disorders.[3] However, despite the biological and medical relevance of glycans, their structural complexity and heterogeneity has hampered the understanding of the molecular basis governing their functions. The relative scarcity of biochemical and analytical tools to decipher the biology that is concealed within their structures is another important obstacle in this direction. For these reasons, the pace of development of glycan-based platforms for diagnostic and therapeutic applications has hitherto been relatively slow. In addition, the intrinsic unfavorable pharmacokinetic properties of carbohydrates have further delayed their exploitation in medicinal chemistry as a source of new drugs.[4] Fortunately, recent advances in the broad field of glycoscience, including novel approaches in carbohydrate chemistry (glycochemistry) and biology (glycobiology), have considerably increased our understanding of the complex correlation between glycan structure and function.[5] However, while improved methods in enzymatic and chemical synthesis of oligosaccharides have been developed over the past years that enabled access to a wide range of complex carbohydrate structures, more widely applicable synthetic strategies are needed to produce larger quantities of glycans for in-depth structure– activity relationships studies. Similarly, the development of new biochemical tools to probe modifications in cell-surface glycosylation related to cancer, inflammation, and infection is necessary to identify unique glycan markers of transformed cells that can be exploited for the discovery of novel diagnostic and therapeutic approaches.[6] Determining the basis of protein–carbohydrate interactions is also important to improve our understanding of glycanmediated molecular recognition processes. In recent years, carbohydrate microarrays have been central in this respect, enabling the rapid analysis of binding events that involve cell-surface oligosaccharides. Microarrays have been widely used for a number of purposes, including the characterization of the sugar binding specificities of many lectins, enzymes and antibodies, detection of specific cells and pathogens (e.g., influenza virus, Leishmania) interacting with glycans, and identifica-
[a] Dr. A. Fernndez-Tejada, Prof. Dr. F. J. CaÇada Chemical and Physical Biology Centro de Investigaciones Biolûgicas (CIB-CSIC) Ramiro de Maeztu 9, 28040 Madrid (Spain) E-mail: [email protected] [b] Prof. Dr. J. Jim¦nez-Barbero CIC bioGUNE: Center for Cooperative Research in Biosciences Bizkaia Technology Park, 48160 Derio (Spain) E-mail: [email protected] [c] Prof. Dr. J. Jim¦nez-Barbero Ikerbasque, Basque Foundation for Science Mara Lûpez de Haro 13, 48009 Bilbao (Spain)
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Viewpoints tion of anti-glycan antibodies associated with a range of diseases such as malaria and cancer. Therefore, it becomes apparent that these carbohydrate array technologies hold great promise for medical applications, not only as diagnostic tools to identify disease-related glycan biomarkers and to detect these markers in patient samples, but also as a useful platform to obtain valuable information for drug discovery purposes as well as for the development of oligosaccharide-based vaccines.[7] Despite the excellent role of glycans as recognition molecules, the exploitation of many pathophysiologically relevant sugar–protein interactions has not yet reached its full potential. Thus, carbohydrates represent a promising but relatively untapped source of new drug targets, as demonstrated by the fact that few carbohydrate-based pharmaceuticals are currently on the market.[8] Beyond the synthetic challenges associated with their structures, one of the main reasons is the suboptimal physicochemical properties of sugars as drug candidates, including their weak monovalent binding affinities, their hydrophilic nature and low tissue permeability, and their short serum half-lives. In addition, another barrier to the full exploitation of the broad potential of complex glycans for the development of effective pharmaceuticals involves overcoming the basic challenges associated with the elucidation of relevant, detailed structure–activity relationships in carbohydrates.[9] This is mainly due to their vast structural heterogeneity and functional complexity, along with the relative scarcity of chemical and analytical tools to manipulate and accurately measure these complex glycan structures. Most of these drawbacks have recently started to be addressed with the rational design of smaller-sized carbohydrate derivatives and mimics that have improved drug-like properties.[10]
The development of such glycomimetics requires extensive knowledge of the conformation and three-dimensional structure of glycans and, more importantly, of the molecular basis of the interaction with their protein receptors (i.e., antibodies, enzymes or lectins). Two major experimental techniques for this atomic-level characterization are NMR spectroscopy and Xray crystallography. The transferred NOE (tr-NOESY) method allows to determine the bound conformation of the sugar at the protein binding site, whereas the saturation transfer difference (STD) NMR gives information on the specific sites of the glycan (ligand epitope) that are in close contact with the receptor.[11] Information gained from these experiments is usually complemented with computational modeling, for example molecular dynamics simulations, to more accurately deduce the bioactive glycan conformation. This knowledge can thus be used for medicinal chemistry and drug discovery efforts in the design and synthesis of new glycomimetics with enhanced binding affinity or inhibitory properties and improved synthetic accessibility. Several successful examples of carbohydrate-based drugs include the influenza virus neuraminidase inhibitors zanamivir and oseltamivir, and the glucosidase inhibitors acarbose and miglitol against diabetes (Figure 1).[12] Another related iminosugar, miglustat, inhibits not only a-glucosidase but also ceramide glucosyltransferase, and is used for the treatment of lysosomal storage disorders (LSDs), namely Gaucher and NiemannPick C diseases.[13] These pathologies are associated with abnormal glycolipid metabolism due to defects in the activity/ trafficking of lysosomal hydrolases, which results in intracellular accumulation of glucosylceramide and glycosphingolipids, respectively. While replacement of the deficient enzyme (glucocerebrosidase in Gaucher patients) and the substrate reduction
Figure 1. Some monosaccharide and oligosaccharide mimics (“glycomimetics”) currently on the market as important examples of approved, small-molecule carbohydrate-based drugs.
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Viewpoints therapy (SRT) have proved to be effective in the treatment of some LSDs, synthetic monosaccharide mimics able to restore the activity of those pathologically mutated glycosidases are already under clinical evaluation and constitute the basis of the chemical chaperoning therapeutic approach.[14] In addition to these monosaccharide-derived glycomimetics, heparin is another blockbuster drug that has tremendously benefited from the power of medicinal chemistry. Structure–activity investigations and extensive understanding of heparin’s mode of action enabled access to a family of low-molecularweight heparins (LMWHs) with improved anticoagulant function. Synthetically defined and therapeutically active carbohydrate mimics have also been developed, by chemical and chemoenzymatic methods, that incorporate the precise heparin oligosaccharide sequence that is known to interact with antithrombin III as the basis of its anticoagulant activity.[15] The first example of such synthetic heparin analogues, the pentasaccharide fondaparinux, exhibited enhanced potency and better safety profile, and has been on the market since 2002 (Figure 1).[16] The heparin full polysaccharide has also been shown to have several other activities and roles in different diseases, such as cancer, infection, and inflammation, which reveals the potential for additional medicinal chemistry studies on the heparin scaffold for the discovery of new therapeutic approaches. Besides free carbohydrates, glycosylated proteins hold great promise for use as pharmaceutical agents. In particular, the modification of intracellular proteins at their serine or threonine residues with O-linked b-N-acetylglucosamine (O-GlcNAc), which is controlled by two enzymes [O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)] is attracting a great deal of interest in the medicinal community because of its altered regulation in Alzheimer’s disease, cancer, and diabetes.[17] Despite the development of some chemical approaches to study this modification, progress in understanding the functional roles of O-GlcNAc in the cell has been slow and more efficient tools are still required.[18] While small molecule regulation of these enzymes is still underdeveloped, the recent discovery by Vocadlo and co-workers of an OGA inhibitor that slows neurodegeneration by increasing O-GlcNAc levels in mouse brain has identified OGA as an important drug target in Alzheimer’s disease.[19] The field of therapeutic glycoproteins constitutes another relevant area for biomedical research and drug development. In these glycoconjugates, the carbohydrate moiety is essential to modulate the properties of the protein, including folding, solubility, stability and biological activity.[20] However, the heterogeneous nature of recombinantly derived glycoproteins complicates the understanding and elucidation of the influence of specific carbohydrates and their sites of glycosylation on the protein therapeutic action. In fact, different glycosylated variants (glycoforms) of a protein drug have distinct physicochemical and biological properties, and given their complexity, detailed structure–function relationships in this type of pharmaceuticals are lacking. As a consequence, recent efforts in the field have focused on the preparation and evaluation of homogeneous glycoproteins that can yield useful structure–activity ChemMedChem 2015, 10, 1291 – 1295
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correlations for advancing our knowledge on the impact of specific sugars on the protein biological function. Given the importance of protein glycosylation, several strategies have been pursued over the past years in an attempt to control this ubiquitous modification to produce tailor-made, fully active glycoprotein drugs. These approaches have been extensively reviewed elsewhere,[21] and include recombinant expression by genetic manipulation of the cell glycosylation machinery, in vivo metabolic oligosaccharide engineering for the incorporation of artificial monosaccharides into natural glycoproteins in the cell, in vitro glycan remodeling using exogenous enzymes, and synthetic chemistry methodologies based on chemoselective ligation protocols and de novo total synthesis. Because the efficacy of protein biopharmaceuticals critically depend on their specific glycoforms, some of the previous strategies have been applied for the production of improved therapeutic glycoproteins, being the blockbuster anti-anemia drug erythropoietin (EPO) and monoclonal antibodies some of the most representative cases. EPO is a 166-amino-acid-long glycoprotein containing four glycosylation sites (three N-linked and one O-linked) that has been widely used in recombinant form (epogen) for the stimulation of red blood cell formation. On the basis of previous studies revealing the key importance of glycosylation (or lack thereof) on EPO pharmacokinetics and in vivo stability, particularly the extent of sialylation on its N-linked sugars, a new version of the drug incorporating additional N-glycosylation sites (darbepoetin) was produced by genetic engineering that possessed improved therapeutic properties, namely prolonged serum half-life and increased potency.[22] To overcome the heterogeneity issues associated with biologically derived material, Danishefsky and co-workers recently accomplished the chemical synthesis of a fully glycosylated and active EPO in homogeneous, pure form.[23] Another example of the therapeutic potential of carbohydrates is their essential role in determining the effector functions and inflammatory activities of monoclonal antibodies. For instance, deglycosylation of immunoglobulin G has been shown to decrease binding to Fc (fragment crystallizable) protein receptors located on the immune cell surface, thereby abrogating antibody effector functions and decreasing inflammation, which has led to the investigation of therapeutic deglycosylated antibodies.[24] Sialylation of the Fc N-glycans also attenuates Fc receptor binding and induces anti-inflammatory effects, while defucosylation causes the opposite effect.[25] Thus, although these complex biomolecules may be viewed as outside of the medicinal chemistry field of action, the previous examples show that by understanding the effect of glycosylation on glycoprotein pharmaceuticals and through rational manipulation of their carbohydrate structures, it is possible to modify their biological functions and maximize their therapeutic potential. Considering the relevance of cell-surface carbohydrates as elements of molecular recognition by the immune system, glycans have also been key actors in this area for the development of oligosaccharide-based vaccines.[26] However, due to the poor immunogenicity of carbohydrates, which mainly elicit
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Viewpoints B-cell-dependent immune responses alone, their covalent attachment to protein carriers, and also co-administration with appropriate adjuvants, has enabled the generation of glycoconjugate vaccines that can elicit more potent, T-cell-mediated responses. Recent advances in carbohydrate chemistry together with the discovery of more efficient synthetic and biochemical tools has facilitated the design and synthesis of several vaccine candidates against infectious diseases produced by bacteria (Haemophilus influenza, Streptococcus pneumoniae, Mycobacterium tuberculosis), and parasites (Plasmodium falciparum, Leishmania).[27] Much research efforts have also been devoted to develop carbohydrate-based anti-HIV vaccines based on the discovery of potent broadly neutralizing antibodies (BnAb) against HIV-1, namely 2G12, and most recently PG9 and PG16. The increasing molecular understanding of the interaction between these BnAbs and the glycan structures on the viral surface has driven the design and synthesis of many oligosaccharide and glycopeptide antigens as epitope mimics for HIV-1 vaccine development.[28] While most of these synthetic derivatives showed good binding affinity for the relevant antibody, all attempts to elicit BnAbs by immunization have failed thus far. Nevertheless, these studies have yielded useful structure–activity relationships for carbohydrate antigenicity and also provided important insights into the glycan specificities for recognition by these antibodies.[29] With this valuable structural information and increasingly better tools in the glycoscience field, it is hoped that medicinal chemistry efforts in the characterization and reconstitution of minimal neutralizing epitopes by chemically defined glycan structures will provide new opportunities for HIV vaccine design. Carbohydrate-based anticancer vaccines have been the subject of intensive research for decades and have gained even greater interest over the last few years. This burgeoning field has been fueled by recent advances in glycobiology and glycochemistry that have contributed to identify an increasing number of tumor-associated carbohydrate antigens (TACAs), which are abnormally expressed in the surface of cancer cells but not in normal cells. Using the tools of chemical synthesis, a number of research groups around the globe have targeted these TACA structures to develop anticancer vaccine candidates.[30] Again, conjugation of the carbohydrate antigens to a protein carrier or another immunogenic delivery system, in combination with a potent immunoadjuvant (e.g., QS-21),[31] has been one of the most common approaches to produce robust T-cell-dependent immune responses. Several forms of TACAs have been explored, either as single or clustered entities, or even as multi-antigenic systems or unnatural, chemically modified variants. After a number of moderately successful two-component self-adjuvanting vaccines, where the B-cell epitope (Tn antigen) was linked to a Pam3Cys lipopeptide serving as an immunostimulant, fully synthetic three-component vaccines have recently been developed. These designs incorporate all the requisite elements necessary for strong immune activation, that is, B-cell and T-cell epitopes as well as a built-in adjuvant. As a significant example, the Boons tripartite vaccine was able to generate potent humoral and cellular responses ChemMedChem 2015, 10, 1291 – 1295
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and to decrease tumor size in mice, which gives some hope for future clinical trials in cancer patients.[32] Several other multicomponent constructs have also resulted in excellent immune responses,[33] which signals the promise of this fully synthetic strategies as potential carbohydrate-based vaccines for cancer immunotherapy. In conclusion, this Viewpoint article hopes to highlight the booming development of glycans at the interface of medicinal chemistry and chemical biology. The remarkable examples outlined herein are intended to illustrate the great promise of carbohydrates, both alone and as main constituents of other glycoconjugates, namely glycoproteins, as the next frontier in pharmaceutical discovery. The fields of glycobiology as well as carbohydrate and glycoprotein synthesis have made a comeback and have a bright future, with novel findings on the physiological roles of glycans and improved chemical and biological tools already appearing on the horizon.
Acknowledgments Generous financial support of the authors’ research is provided by the European Commission (Marie Curie International Outgoing Fellowship to A.F-T.), and the Spanish Ministry of Economy and Competitiveness (MINECO) (grant CTQ2012-32025). Keywords: carbohydrates · chemical glycobiology · drug discovery · glycans · medicinal chemistry · oligosaccharide synthesis [1] Glycoscience: Chemistry and Chemical Biology, 2nd ed. (Eds.: B. O. FraserReid, K. Tatsuta, J. Thiem, G. L. Cot¦, S. Flitsch, Y. Ito, H. Kondo, S.-i. Nishimura, B.Yu), Springer, Berlin, 2009. [2] Essentials of Glycobiology, 2nd ed. (Eds.: A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart, M. E. Etzler), Cold Spring Harbor Laboratory Press, New York, 2009. [3] D. Sols, N. V. Bovin, A. P. Davis, J. Jim¦nez-Barbero, A. Romero, R. Roy, K. Smetana, H.-J. Gabius, Biochim. Biophys. Acta 2015, 1850, 186 – 235. [4] L. Cipolla, F. Peri, Mini-Rev. Med. Chem. 2011, 11, 39 – 54. [5] J. E. Hudak, C. R. Bertozzi, Chem. Biol. 2014, 21, 16 – 37. [6] a) L. X. Wang, B. G. Davis, Chem. Sci. 2013, 4, 3381 – 3394; b) D. H. Dube, C. R. Bertozzi, Nat. Rev. Drug Discovery 2005, 4, 477 – 488. [7] a) S. Park, J. C. Gildersleeve, O. Blixt, I. Shin, Chem. Soc. Rev. 2013, 42, 4310 – 4326; b) C. D. Rillahan, J. C. Paulson, Annu. Rev. Biochem. 2011, 80, 797 – 823. [8] M. C. Galan, D. Benito-Alfonso, G. M. Watt, Org. Biomol. Chem. 2011, 9, 3598 – 3610. [9] Z. Shriver, S. Raguram, R. Sasisekharan, Nat. Rev. Drug Discovery 2004, 3, 863 – 873. [10] J. L. Magnani, B. Ernst, Discov. Med. 2009, 8, 247 – 252. [11] a) L. Unione, S. Galante, D. Diaz, F. J. CaÇada, J. Jim¦nez-Barbero, Med. Chem. Commun. 2014, 5, 1280 – 1289; b) V. Roldûs, F. J. CaÇada, J. Jim¦nez-Barbero, ChemBioChem 2011, 12, 990 – 1005. [12] B. Ernst, J. L. Magnani, Nat. Rev. Drug Discovery 2009, 8, 661 – 677. [13] a) T. D. Butters, R. A. Dwek, F. M. Platt, Glycobiology 2005, 15, 43R – 52R; b) T. D. Butters, R. A. Dwek, F. M. Platt, Curr. Top. Med. Chem. 2003, 3, 561 – 574. [14] a) Y. Suzuki, Brain Dev. 2013, 35, 515 – 523; b) L. Cipolla, A. C. Araffljo, D. Bini, L. Gabrielli, L. Russo, N. Shaikh, Expert Opin. Drug Discovery 2010, 5, 721 – 737. [15] R. J. Linhardt, J. Liu, Curr. Opin. Pharmacol. 2012, 12, 217 – 219. [16] M. Petitou, C. A. van Boeckel, Angew. Chem. Int. Ed. 2004, 43, 3118 – 3133; Angew. Chem. 2004, 116, 3180 – 3196.
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