From carbohydrate leads to glycomimetic drugs
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Nature Reviews Drug Discovery | AOP, published online 24 July 2009; doi:10.1038/nrd2852 REVIEWS
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.
Glycocalyx
All cells are coated with complex carbohydrates called glycanbinding epitopes. These discoveries have led to
Literally meaning ‘sugar coat’, glycans, which form a layer known as the glycocalyx, a renaissance in glycobiology. They also provide a con
it is the outer component of ranging from 10 to 100 nm in thickness1,2. Glycans are tinuous supply of carbohydraterelated targets for the
a cell surface and contains a present in many different molecular forms, including structurebased design of new chemical entities that
network of polysaccharides
glycoproteins, proteoglycans, glycolipids and glycophos mimic bioactive carbohydrates, and form a novel class
and complex carbohydrates.
phatidylinositollinked proteins. Their broad diversity of therapeutics.
Anomeric centre originates from their assembly from monosaccharide
The centre of chirality of a building blocks, which can be linked to each other at Carbohydrate and carbohydrate-derived drugs
glycan that is generated by
various positions on their pyranose or furanose rings. Although carbohydrates play an important part in a vast
hemiacetal ring closure.
Each ring can establish several linkages, giving rise to array of biological processes, carbohydrate and carbo
branched structures. Finally, the structural complexity hydratederived drugs cover only a limited area of the
of glycans is further increased by the possibility of α and world of therapeutics (FIG. 1). Many pathophysiologically
βisomers at the anomeric centre. important carbohydrate–protein interactions have yet to
This dense structural information is decoded by be exploited as a source of new drug targets. One reason
carbohydratebinding proteins, which are involved in might be the pharmacokinetic drawbacks that are inher
important physiological and pathophysiological events. ently linked to carbohydrates. As a result of their high
The need for an integrated approach to decipher the polarity, they are unable to cross passively through the
*Institute of Molecular structure–activity relationships (SARs) between glycans enterocyte layer in the small intestine — a prerequisite
Pharmacy, University of and their protein receptors has led to the establishment for oral availability. In addition, once systemically avail
Basel, Klingelbergstrasse 50, of interdisciplinary collaborative efforts in the United able by parenteral administration, carbohydrates suffer
CH‑4056 Basel, Switzerland.
States (Consortium for Functional Glycomics; see from fast renal excretion.
‡
GlycoMimetics, Inc.,
101 Orchard Ridge Drive, Further information), Europe (EuroCarb; see Further When interactions with blood plasma components
Gaithersburg, Maryland information) and Japan (Human Disease Glycomics/ are possible, the plasma halflife that is required for a
20878, USA. Proteome Initiative; see Further information). successful therapeutic application can be achieved.
Correspondence to B.E. Currently, over 80 carbohydratebinding proteins Prominent examples are the lowmolecularweight
e‑mail:
beat.ernst@unibas.ch
have been identified. The binding specificities for heparins, derived from animal tissue, and fondaparinux 3
doi:10.1038/nrd2852 many of them have been elucidated, and others are (Arixtra; GlaxoSmithKline), which are used as anti
Published online 24 July 2009 being screened on large glycoarrays to determine their coagulants. In other cases — such as the inhibition of
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REVIEWS
O OH
OH O
HO OH S NH2
CO2H HO
O O HO
AcHN CO2Et OH O HO HO
HN O HN
AcHN HO NnBu O O OH
NH HO
H3N
H2N H2PO4 OH O O OH
Zanamivir (Relenza)7 Oseltamivir phosphate (Tamiflu)9 Miglustat (Zavesca)226 Topiramate (Topamax)227 Voglibose (Glustat)4
HO OSO3Na
HO O
HO HO OSO3Na
HO OH
HO HN HO O NaO2CH2 OSO3Na
HN O O
OH O O O OSO3Na
HO OH NaO3S O
OH HO
NaO2CH2 NaO3S HN HO O
O O O O HO
NaO3S
HO N HO OH HN
OCH3
HO HO NaO3S
O O Fondaparinux (Arixtra)10
OH HO
OH OH
Miglitol (Glyset)5 Acarbose (Glucobay)6
NaO3S
OR OSO3Na
NaO2CH2 O
HO O O OH
O
HO O HO R2
NaO2CH2 OH
OSO3Na R1HN O
O O OH
OH HO O O OSO3Na HO
NaO2C O O O OSO3Na R3 OR n
O O HO O O
O NaO3S HN HO O
HO O O O HO n = 3–20; R = H or SO3Na; R1 = SO3Na or Ac
NaO3S
OH NHAc NaO2CH2 AcHN R2 = H, R3 = CO2Na or R2 = CO2Na, R3 = H
n O
n
Sodium hyaluronate (Orthovisc)228 Ardeparin sodium (Normiflo)229 Dalteparin sodium (Fragmin)229
(1/2Ca)O3S O
OR OSO3(1/2Ca) OR1
(1/2Ca)O2C O OR1 O
O O OH
HO O HO O O OR1 R4 OR1 OH
HO O RO R2 HO R4 OR1
O O HO O
R1HN O R2HN HO O R=
O O OH O O
HO CO2Na O HO NHSO3Na
R2HN R3
R3 OR n R3 O R
n 15–25%, n = 0–20;
R = H; 75–85%, n = 1–21
R = H or SO3(1/2Ca); R1 = H, SO3(1/2Ca) or Ac R1 = H, SO3Na; R2 = SO3Na, Ac;
R2 = H, R3 = CO2(1/2Ca) or R2 = CO2(1/2Ca), R3 = H R3 = H, R4 = CO2Na
or R3 = CO2Na, R4 = H
Nadroparin calcium (Fraxiparin)229 Enoxaparin (Lovenox)229
Figure 1 | carbohydrate and carbohydrate-derived drugs. Structures of currently approved drugs (trade name
in brackets). These include glycosidase inhibitors that prevent the digestion of carbohydrates for the treatment
of diabetes (voglibose4, miglitol5 and acarbose6) and the prevention of influenza virus infections (zanamivir
Nature Reviews | Drugand
7
Discovery
oseltamivir9); and sulphated glycosaminoglycans, which function as anticoagulants by binding to antithrombin III for
the treatment of thrombosis (fondaparinux , dalteparin , ardeparin , nardoparin and enoxaparin ). In addition,
3 161 161 161 161
carbohydrate-derived drugs are used to treat Gaucher’s disease (miglustat162), epilepsy (topiramate163) and
osteoarthritis (sodium hyaluronate164).
αglycosidases in the brush border of the small intes Glycodrugs in preclinical and clinical evaluation
tine for the treatment of diabetes (by voglibose4 (basen/ Carbohydratebinding proteins are broadly classified
Glustat/volix; Takeda), miglitol5 (Glyset; Pfizer) and into lectins10 and sulphated glycosaminoglycan (SGAG)
acarbose6 (Glucobay/Prandase/Precose; bayer)) or the binding proteins11,12. There are two categories of lectins
Lectin inhibition of viral neuraminidases in the pharyngeal present in vertebrates: the families of intra cellular
A carbohydrate-binding mucosa (by zanamivir 7 (Relenza; GlaxoSmithKline)) — lectins (for example, calnexin, ltype and Ptype lectins),
protein which is highly specific
oral availability is not required. which bind core oligosaccharide structures and are
for sugar moieties and typically
plays a part in biological The paradigm of a glycomimetic drug in the classical involved in glycoprotein processing and quality control,
recognition phenomena. sense is oseltamivir (Tamiflu; Gilead/Roche). Starting and the families of extracellular lectins (for example,
from a carbohydrate lead, drug likeness was achieved galectins, Ctype, Itype and Rtype lectins), which recog
Sulphated by systematically eliminating polar groups and meta nize terminal carbohydrate epitopes of other cells and
glycosaminoglycan
A long polysaccharide chain
bolic ‘soft spots’8 that were not required for affinity. pathogens. Extracellular lectins account for most of the
consisting of repeating Finally, by designing a prodrug, oral availability became molecular targets that are being investigated in current
sulphated dissacharide units. possible9. drug discovery programmes.
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REVIEWS
by contrast, SGAGbinding proteins are heterogen To functionally bind sialyl lea/x in vivo, both P and
eous and difficult to classify 11,12. Their ability to recognize lselectins require additional interactions with negatively
SGAGs arises from clusters of cationic amino acids on charged sulphate groups, either on the carbohydrate chain
unrelated proteins that confer the ability to recognize itself or on an adjacent peptide sequence. Eselectin has
anionic structural motifs in extended SGAG chains. no such requirement and can functionally bind sialyl
Typically, various SGAGbinding proteins interact with lea/x in glycolipids25 and glycoproteins26.
each SGAG with different affinities, and only a few SGAG The involvement of negatively charged groups, such
sequences are exclusively recognized by a single SGAG as sulphates and carboxylates, in the binding of l and
binding protein. Pselectin has led to one of the major pitfalls in designing
Here, we present the most promising drug candidates smallmolecule inhibitors for the selectins. A wide range
from the lectin families: selectins and dendritic cell of structurally diverse, negatively charged molecules has
specific ICAM3grabbing nonintegrin 1 (DCSIGn; also been reported to bind P and lselectins. These include
known as CD209) from the Ctype lectin family, myelin sulphatides27, heparins28, fucoidan29, sulphated dextran30,
associated glycoprotein (MAG; also known as sialic acid chondroitin sulphate31, dermatan sulphate32, tyrosine
binding immunoglobulinlike lectin 4A (Siglec 4A)) as sulphates33, sulphated hyaluronic acid34 and sulpho
an example of an Itype lectin, and PAI galactophilic galabiose35. Such a range of molecules suggests that their
lectin (PAIl), fucosebinding lectin PAIIl and minor inhibitory activity is due to nonspecific negativecharge
component of type 1 fimbriae (FimH) as representatives interactions. In fact, a cautionary publication36 described
of bacterial lectins. potent Pselectin activity found in trace contaminants of
polyanions from ion exchange media used in the prepa
C-type lectins ration samples. Thus, the specificity of smallmolecule,
The hallmark of Ctype lectins is the involvement of Ca2+ highly charged selectin antagonists that inhibit P and
in the binding of glycans to their carbohydrate recognition l but not Eselectin must be carefully evaluated.
domain (CRD). They have a wide range of biological func In diseases in which cell adhesion, extravasation of
tions, such as intercellular adhesion, serum glycoprotein cells from the bloodstream or the migration of specific
removal and pathogen recognition. lymphocytes has been implicated in the pathology,
selectins present an attractive therapeutic target. For
Selectins. These are perhaps the most intensely studied example, E and Pselectins have been shown to mediate
mammalian carbohydratebinding proteins. First dis the acute adhesion and aggregation of leukocytes and
covered in 1989 (REFS 13–15), their functions as adhesion erythrocytes during a vasoocclusive crisis in a mouse
molecules are well understood16. The family consists model of sickle cell disease37,38. Furthermore, aberrant
of three members: Eselectin (also known as CD62E), extravasation of cells from the bloodstream is the hall
Pselectin (also known as CD62P) and lselectin (also mark of many inflammatory diseases (such as asthma,
known as CD62l). They are composed of a Ca2+ colitis, arthritis and psoriasis) and cancer. Tumour cells
dependent CRD, an epidermal growth factor (EGF) that extravasate out of the bloodstream use the selec
domain, various short complementlike consensus repeats, tin pathway to metastasize. Many solid tumours and
a single transmembrane domain and an intracellular tail. adenocarcinomas, such as gastrointestinal39, pancreatic40,
Although carbohydrates bind to a receptor site within the breast41, lung 42 and prostate43 cancers, express high levels
CRD, the neighbouring EGF domain influences binding of slex and slea. Expression of these selectin ligands
affinity and specificity 17. on the tumour cells of patients with gastric and colon
The three selectins have overlapping and distinct cancers44 is significantly correlated with poor survival22.
expression patterns, both temporally and spatially. Cimetidine (Tagamet; GlaxoSmithKline), a histamine
Eselectin is expressed on endothelial cells by de novo pro receptor antagonist that also suppresses vascular expres
tein synthesis 2–4 hours after stimulation by inflammatory sion of Eselectin, markedly and specifically improved
mediators, such as interleukin 1β and tumour necrosis survival of highrisk patients identified by tumour
factorα. Pselectin is expressed on activated platelets and expression of slea and slex (REF. 45), further supporting
is also stored in Weibel–Palade bodies in endothelial cells, the usefulness of selectins as therapeutic targets for
which fuse to the cell surface on activation, leading to the cancer.
expression of Pselectin within minutes. lselectin is con Selectins and their ligands have also been reported
stitutively expressed by most leukocytes and plays a major to play key parts in the dissemination of haematological
Carbohydrate recognition part in homing and trafficking of lymphocytes through cancers46 and the homing of leukaemic stem cells to
domain the blood and lymphatic systems. microdomains within the bone marrow 47. Eselectin is
The three-dimensional All three selectins bind a common carbohydrate constitutively expressed in the bone marrow48 and binds
domain in a lectin that
domain shared by sialyl Lea/x (sialyl lewisa (slea) and sialyl carbohydrate ligands that are found on leukaemic stem
binds carbohydrate.
lewisx (slex))18. Interestingly, both of these carbohydrate cells. Once adherent to these microdomains in the bone
Lea/x sequences were originally discovered as cancerassociated marrow, leukaemic cells become quiescent and less sus
A common three-dimensional antigens19–21 and are prognostic indicators of metastatic dis ceptible to killing by antiproliferative chemotherapy
structural domain shared by ease22. Tumour cells coated with these carbohydrate chains drugs such as cytosine arabinoside49. Potent selectin
the carbohydrate structures
Lewisa (Galβ(1–3)[Fuc(α1–4)]
are recognized as migrating leukocytes, allowing them to antagonists present new therapeutic opportunities for
GlcNAc) and Lewisx (Galβ(1–4) escape the bloodstream and metastasize to other organs and treating these diseases. by preventing sequestration
[Fuc(α1–3)]GlcNAc). tissues, such as the lymph nodes and bone marrow23,24. of leukaemic cells in the bone marrow and keeping
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them in circulation, combination therapy with selectin To improve the affinity and pharmacokinetic properties
antagonists is likely to make the cells more susceptible of these naturally occuring antagonists, glycomimetics of
to chemotherapy. Some examples of glycomimetic, small both types of ligands have been synthesized.
molecule antagonists of the selectins are presented in Highdensity arrays of unbranched Manα(12)Man
TABLE 1. terminated oligosaccharides bind to DCSIGn almost
as effectively as the entire Man9 oligosaccharide (REF. 68).
DC-SIGN. Mucosal surfaces present barriers to the Therefore, the nonreducing end Manα(12)Man frag
environment that are potentially susceptible to infec ment of Man9 was suggested to play a crucial part in
tion. Migrating dendritic cells guard mucosal surfaces, DCSIGn recognition. To mimic 1,2mannobiose,
capturing microorganisms and presenting processed one hexose moiety was replaced by a cyclohexanediol
antigens to activated T cells, thereby inducing an derivative, leading to the pseudo1,2mannobioside
immune response against the invading pathogens. by compound 1 (FIG. 2), which had a threefold greater
screening a library of dendritic cellspecific mono affinity for DCSIGn than did 1,2mannobiose (half
clonal antibodies that inhibit binding to intercellular maximal inhibitory concentration (IC50) = 0.62 mM and
adhesion molecule 3 (ICAM3; an adhesion molecule 1.91 mM, respectively)69. Furthermore, in infection
that activates T cells), a single cell surface protein was studies using an in vivo model of Ebola infection,
discovered: DCSIGn50. the glycomimetic compound 1 inhibited infection of
The aminoacid sequence of DCSIGn is identical to DCSIGnexpressing Jurkat cells more efficiently than
a previously described HIv glycoprotein 120 (gp120) the corresponding natural disaccharide. Although the
binding Ctype lectin51,52. DCSIGn that is expressed inhibitory concentration in these experiments was in
on patrolling dendritic cells in the mucosa binds to the millimolar range, compound 1 might be useful in
carbohydrate structures on the gp120 protein coat the preparation of highaffinity multivalent antagonists.
of HIv, which is the initial entry port of HIv to the host. Such an approach is encouraged by the strong inhibitory
HIv particles bound to DCSIGn on the surface of den effects of multivalent antagonists on DCSIGn bind
dritic cells are protected from destruction in the blood ing, as observed for dendritic mannose conjugates70 or
and migrate to the lymph nodes where they transinfect oligolysinebased oligosaccharide clusters71.
T cells through the CD4–CCR5 (CCchemokine recep Similarly, αfucosylamine linked to 2aminocyclo
tor 5) complex on the T cell surface51. The binding spe hexanecarboxylic acid (compound 2) mimics lewisx
cificity of DCSIGn is for fucose and mannose residues, trisaccharide and inhibits DCSIGn with a twofold
with higher affinity and specificity for the fucose linkage greater potency (IC50 = 0.35 mM and 0.8 mM, respec
in lea/xtype oligosaccharide structures. Formation of the tively)72. These binding affinities are too weak for these
active structure and binding of DCSIGn occurs in a compounds to have any therapeutic promise; however,
Ca2+dependent manner52,53. when the oligosaccharides are displayed on large multi
In addition to HIv, various other pathogens — such valent dendrimers, activity is greatly improved and bio
as the hepatitis C virus54, Dengue virus55, Ebola virus56, logical activity can be shown in vitro71. Although such
Marburg virus57, coronavirus (which causes severe acute large multivalent presentations of carbohydrates or
respiratory syndrome)58 and cytomegalovirus59, as well mimics thereof are a relatively simple means to increase
as bacteria such as Mycobacterium tuberculosis 60 and activity, they pose a pharmaceutical challenge in terms
Helicobacter pylori 52 and yeast (Candida albicans) — of routes of administration and possible side effects, such
exploit DCSIGn to infect their host. More recently, as unwanted immune responses.
even parasites such as Leishmania spp.61 and Schistosoma A classical approach to discovering DCSIGn antago
mansoni 62 have also been shown to bind DCSIGn. nists was successfully demonstrated by screening large
The fact that different pathogens have capitalized on libraries of small molecules in an automated assay
this infection strategy makes DCSIGn an interesting format. by screening over 35,000 compounds, 7 hits
target for therapeutic intervention. In a study on the with IC50 values in the low micromolar range were
Glycomimetic
A molecular mimic of a binding and transfer of HIv in human rectal mucosa identified, such as compound 3 and compound 4
functional carbohydrate cells, more than 90% of bound virus was bound to cells (REF. 73). Interestingly, the structures of these hits bear
that has improved affinity expressing DCSIGn, although these cells represented no resemblance to the native carbohydrate ligands of
for its target and drug-like only 1–5% of the total mucosal mononuclear cells. oligomannose or the lewis epitopes and do not con
pharmacokinetic properties.
Furthermore, DCSIGnspecific antibodies blocked tain functional groups to interact with Ca2+ in the CRD.
Dendrimer more than 90% of HIv binding 63. Other studies have Their inhibitory activity could be caused by binding to
A large, synthetically produced shown that multivalent glycoconjugates of lewis x other domains on DCSIGn, leading to an allosteric
polymer in which the atoms are or dmannose prevented the attachment of Ebola or effect.
arranged in many branches
herpes virus to dendritic cells through DCSIGn and
and subbranches radiating out
from a central core. thus prevented the subsequent infection of immune I-type lectins
cells64–66. Itype lectins are a family of carbohydratebinding
Allosteric effect Glycomimetic compounds that inhibit DCSIGn are proteins in the immunoglobulin superfamily, and
An effect that is exerted on a based on two lead structures. The first are highmannose include Siglecs74. The Siglecs function as cell signalling
protein by the binding of an
effector molecule at a site
oligosaccharides and the second is lfucose as part of a coreceptors and are primarily expressed on leukocytes
other than the protein’s active lewis epitope67. These determinants are synthesized by that mediate acquired and innate immune functions.
site or binding site. pathogens to camouflage their appearance as host tissue. The cytoplasmic domains of most Siglecs contain
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immunoreceptor tyrosinebased inhibitory motifs, regulatory elements enable Siglecs to carry out unique
which are characteristic of accessory proteins that roles at the cell surface. Siglecs can be broadly divided
regulate transmembrane signalling and endocytosis of into an evolutionarily conserved group (Siglec 1 (also
cell surface receptor proteins. The diverse specificity known as sialoadhesin), Siglec 2 (also known as CD22)
for their sialoside ligands and variable cytoplasmic and Siglec 4 (also known as MAG)) and a Siglec 3related
Table 1 | Small-molecule selectin antagonists in preclinical and clinical trials
Name and structure specificity Disease institution status refs
OH
HO CO2H OH HO OH E-, P- and Cardio- Cytel Stopped 179
OH OH
O O
L-selectin vascular injury
AcHN O O O
O O O OCH3
HO NHAc OH
HO O
OH OH
OH
Cylexin (CY-1503) HO
E-, P- and Asthma and Revotar Phase IIa 180
HO2C (CH2)6 CO2H L-selectin psoriasis
O O
OH OH
O O
OH OH
OH OH
HO HO
Bimosiamose (TBC-1269)
CH3NH O E-, P- and Allergic Nippon Preclinical 181
C O C14H29 L-selectin dermatitis Organon
H
H N
N NH2 O2C N C14H29
H
O
NH2 OH O
HO
O
O2C NH3
OJ-R9188 OH
CO2H
H E-, P- and Sickle cell Glyco- Phase I 182
N
OBz O L-selectin crisis Mimetics
H O O NH
O O
O O
NHO
HO O H O
OH OH N O NH SO3H
O
OH
HO NH
GMI-1070 O
HO3S SO3H
CO2H P-selectin Athero- Wyeth Phase I 183
OH Cl thrombotic
and venous
thrombotic
N
diseases
PSI-697
OH E-, P- and Metastatic Kanebo Unknown 184
OH C14H29 L-selectin cancer
NaO3SO O O
O O O
C14H29
OH
HO O
OH OH
OH
HO GSC-150
E- and Psoriasis Bayer Preclinical 185
O
P-selectin
OH O OH O O OH O OH
O
Efomycin M
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a b MAG. 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 MAG78. 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,
Galβ(1-4)[Fucα(1-3)]GlcNAcβ(1-3)Gal Manα(1-6)[Manα(1-3)]Manα(1-6)Man
which results in growth cone collapse79.
c The RhoA–ROCK inhibitory cascade can also be
HO
OH
HO
OH
HO
OH triggered by a complex formed by MAG, brain ganglio
HO
HO
O
OH HO
HO
O sides (especially GM1b, GD1a, GT1b, GT1β and
O
HO HO OH NHAc
CH3OOC O
GQ1bα)80 and nGFR81. Although the exact biological
O OH
HO O O
O
O
CH3OOC role of the MAG–ganglioside interaction has yet to be
HO HO O
OH
resolved, in some systems inhibition of axon regenera
O
NH2
OH O
NH2
tion by MAG could be completely reversed by sialidase
Manα(1-2)Man Lewis X 1 treatment, suggesting that sialidated glycans are the
IC50: 1.91 mM IC50: 0.8 mM IC50: 0.62 mM main axonal ligands of MAG82. SAR studies83–85 have
revealed that the terminal tetrasaccharide epitope
HO H
neu5Acα(2→3)Galβ(1→3)[neu5Acα(2→6)]GalNAc
OH
N
N
S
of GQ1bα shows superior binding to MAG compared
OH
O
OCH3 N
with the terminal trisaccharide epitope, which is present
HO
HN
3 N
H
O in GD1a and GT1b, for example86. Further refinements
O IC50: 2 µM
COOCH3 O of the SAR profile have led to the identification of MAG
HO CONH
antagonists that have improved affinities and, at least
in some cases, remarkably simple structures (FIG. 3).
N H
2 4 S S N However, owing to the use of different assay formats, it
IC50: 0.35 mM IC50: 1.6 µM has been difficult to compare the reported affinities of
N N N O
H these compounds for various ligands.
Figure 2 | Ligands of dendritic cell-specific icAM3-grabbing non-integrin 1 Overall, starting from the lowaffinity tetrasaccharide
(Dc-sigN). DC-SIGN co-crystallized with the natural epitopes Galβ(1-4)[Fucα(1-3)] lead structure compound 6 (REF. 87), lowmolecular
GlcNAcβ(1-3)Gal (Protein Data Bank code 1SL5) (a) and Manα(1-6)[Manα(1-3)]Manα(1-6)
Nature Reviews | Drug Discovery mass MAG antagonists with nanomolar affinity and
Man (PDB code 1SL4) (b). The protein backbone is depicted in ribbon style, excellent stability in the spinal cord fluid have been
carbohydrates are shown in ball and stick style and the grey sphere is Ca2+. Part c shows identified (S. Mesch, D. Moser, A. vedani, b. Cutting,
the structures of DC-SIGN antagonists. The glycomimetics compound 1 (REF. 69) and M. Wittwer, H. Gäthje, S. Shelke, D. Strasser, O. Schwardt,
compound 2 (REF. 72) have only a slightly improved affinity compared with the natural S. Kelm and b. Ernst, unpublished observations). The
ligands Manα(1-2)Man68 and Lewisx (REF. 72), whereas the non-carbohydrate antagonists
high correlation between the degree of neurite out
compound 3 and compound 4 have half-maximal inhibitory concentration (IC50) values
in the low micromolar range73.
growth and the binding affinities of these antagonists
further validates MAG as a therapeutic target and sug
gests that potent glycan inhibitors of MAG have the
potential to enhance axon regeneration88.
group (Siglec 3 and Siglecs 5–13). The evolutionarily
conserved group shows selective binding properties: Bacterial and viral lectins
Siglec 1 and MAG preferentially bind α(23)linked For colonization and subsequent development of an
Nacetylneuraminic acid (neu5Ac) and Siglec 2 is highly infectious disease, enteric, oral and respiratory bacteria
specific for α(26)linked neu5Ac. by contrast, members require adhesion to the host’s tissue. This grants them a
of the Siglec 3related group are more promiscuous in their substantially greater resistance to clearance and killing by
binding, often recognizing more than one presentation immune factors, bacteriolytic enzymes and antibiotics. In
of neu5Ac. addition, such bacteria are better able to acquire nutrients,
The most comprehensively characterized Siglecs are further enhancing their ability to survive and infect the
Siglec 2, a regulatory protein that prevents the over host. Therefore, antiadhesive drugs that prevent the adhe
Growth cone activation of the immune system and the development sion of pathogens to host tissues may offer a novel strat
A dynamic extension at the
tips of axons that is supported
of autoimmune diseases, and MAG, a protein that blocks egy to fight infectious diseases89. The alarming increase
by actin and grows towards regeneration of the central nervous system (CnS) after in drugresistant bacterial pathogens makes a search for
synaptic targets. injury 75. new approaches to fight bacterial infections essential90.
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a Oligodendrocyte b HO
Nogo 66
MAG MOG MAG
NHAc
HO
HO OH
O
–S–S– –S–S– –S–S–
–S–S– –S–S– –S–S–
HO2C
O OH OH
OH AcHN
O HO
HO OH OH
––S–S–
––S–S–
O
S–
S–
O OH
S–
S–
NGFR NGFR AcHN HO
O CO2H
GD1a HO
O O
RTN4R GT1b HO OH HO AcHN
O AcHN
GQ1bα O
HO2C
OH O
OH
O
OH O
HO2C
OH OHO OH
O O
GQ1bα OH
Neuron
HN OH
Activation Growth cone
of RhoA collapse
O
OSO3H C16H34 C13H27
HO OH HO OH HO
HO
OH AcHN O O
AcHN O O O O OH
OH OH
HO OH AcHN OH
HO O CO2H CO2H O HO
OH
HO3SO O O C13H27
O
O 5 OH HN C17H35
HO OH HO OH
HO
AcHN O O O
O O O OSE HO OH
HO OH OH NHAc OH
HO2C AcHN
OH
6 HO O CO2H
Kd: 180 µM
O
O HO OH HO
HO HO
HO OH AcHN O O
AcHN O O O O
O O
AcHN OH OH
OH N OH
HO CO2H CO2H
OH H OH
HO O CO2H 8 9
RIP: 0.41 Kd: 2.8 µM
O
HO OH HO
HO O
HO HO
AcHN O O
O O O AcHN FH2C N
O O OCH3 O H O O
HO OH OH AcHN OH F
HO2C O N CO2H N OH
OH H OH H CO2H
OH F
AcHN COOCH3
7 10 11
Cl
IC50: 300 nM Kd: 143 µM Kd: 500 nM
Figure 3 | Myelin-associated glycoprotein (MAg) antagonists. a | MAG, nogo 66 and myelin oligodendrocyte
glycoprotein (MOG) bind to the reticulon 4 receptor (RTN4R; also known as the nogo receptor). The inhibitory signal is
transduced into the cytosol of the neuron through the co-receptor NGFR (nerve growth factorNature receptor; also Discovery
Reviews | Drug 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 cytosol79. b | GQ1bα is the brain ganglioside with the highest affinity for
MAG80; replacement of its inner sialic acids by sulphates (to produce compound 5) led to a fourfold increase in
affinity165. 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 established88. Further modifications involved
the replacement of the Galβ(1-3)GalNAc core (to produce compound 8 (REF. 166)) or the α(2-6)-linked Neu5Ac (to
produce compound 9 (REF. 135)). Following studies on compound 10 (REF. 167), numerous Neu5Ac derivatives168,169,
for example, compound 11, with up to nanomolar affinities have been synthesized. Affinity data of the different
compounds should be compared with caution as they were obtained from different assays. IC50, half-maximal
inhibitory concentration; Kd, dissociation constant; RIP, relative inhibitory potency.
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because antiadhesive agents are not bactericidal, they are detected as a result of the higher flexibility of Fucα(14)
less likely to promote the propagation of resistant strains GlcNAc compared with lewisa. However, additional
than bactericidal agents, such as antibiotics. enthalpic interactions that originate from a network of
The carbohydrate epitopes on the surface of host cells hydrogen bonds compensate for this entropic penalty 101.
that are used by bacteria and viruses for colonization and A further simplification of the PAIIl antagonists was
infection (TABLE 2) are the starting point of the search for achieved when αlfucosides bearing heterocyclic sub
glycomimetic entry inhibitors. stituents as aglycons were synthesized. Surprisingly, some
A challenge of antiadhesion therapy is that most candidates — for example, compound 13 —have a similar
pathogens possess genes encoding several types of potency to lewisa (REF. 99).
adhesins, so that, during the infection process, they may Oligovalent forms of the Fucα(14)GlcNAc epitope,
express more than one of these adhesins. Glycomimetic such as compound 14 (REF. 102), exhibit increased activity
antagonists that are designed to inhibit multiple adhesins compared with monovalent forms; however, in most
are feasible to develop, and examples are described below cases, this effect was only modest on a per saccharide
for Pseudomonas aeruginosa. basis. To date, multivalency has only been explored
with dendrimers that present lfucose, which show an
P. aeruginosa virulence factors (PA-IL and PA-IIL). increase in affinity of up to a factor of 20 on a per saccha
P. aeruginosa can be part of the normal flora in healthy ride basis103. Finally, to prevent adhesion of P. aeruginosa
adults but becomes a deadly pathogen in individuals who mediated simultaneously by the PAIl and PAIIl lectins,
are immunocompromised, patients with cystic fibrosis heterobifunctional ligands that present both dgalactose
and hospitalized, critically ill patients. An increasing and lfucose in an oligovalent array (as in compound 15
percentage of P. aeruginosa infections are antibiotic (REF. 104)) or as a smallmolecule glycomimetic (as in
resistant. compound 16 (REF. 105)) have been constructed. In a
For its adhesion to host cells, the pathogen expresses study to determine the efficacy of compound 16 in
lectins such as PAIl and PAIIl91. These lectins are mice surgically stressed by 30% hepatectomy, 60% of
virulence factors under quorum sensing control and the control group died 48 hours after acute infection
are, by themselves, cytotoxic to primary epithelial cells with P. aeruginosa, whereas 100% of mice treated with
in culture92. At low concentrations, they inhibit ciliary compound 16 survived105.
beating of epithelial cells in explants of nasal polyps93.
Inhibition can be completely reversed by treatment with FimH. Urinary tract infections (UTIs) are among the
the carbohydrate ligand of the lectin. Thus, 24 hours most prevalent inflammatory diseases that are caused
after addition of fucose, ciliary beating returns to normal by pathogens106,107. The predominant pathogen in UTIs
frequency 94. is uropathogenic Escherichia coli (UPEC), which causes
PAIl and PAIIl are tetrameric lectins that require more than 80% of all infections in otherwise healthy
Ca2+ for carbohydrate binding. The crystal structures of people (uncomplicated UTI). In healthy individuals,
both lectins complexed with their carbohydrate ligands most uropathogens originate from the rectal microbiota
have been resolved (FIG. 4). PAIl preferentially binds and enter the normally sterile urinary bladder through
to terminal αlinked dgalactose in the presence of the urethra, where they trigger the infection (cystitis).
one Ca2+ ion, whereas PAIIl binds with an unusually Once in the urinary tract, bacteria attach to the urinary
strong micromolar affinity to lfucose and requires two tract epithelium through fimbrial adhesion molecules to
Ca2+ ions95,96. PAIl and PAIIl are soluble intracellu avoid the host’s defence mechanisms. Once bound, the
Adhesin
lar lectins. However, once released from the cells, these bacteria are presumably internalized in an active process
A protein produced by many lectins cause bacteria to adhere to host tissue — a process that is similar to phagocytosis108.
bacteria to effectively adhere that can be reversed by incubation with dgalactose and Uncomplicated UTI can be effectively treated with
to host surfaces. dmannose, respectively 97. oral antibiotics such as fluoroquinolones, cotrimoxazol
The native carbohydrate inhibitors of PAIl and or amoxicillin and clavanulate, depending on the sus
Quorum sensing
A type of decision-making PAIIl, dgalactose and lfucose, were successfully used ceptibility of the pathogen involved. However, recurrent
process used by bacteria to to treat a tobramycinresistant P. aeruginosa infection in infections and subsequent antibiotic exposure can result
coordinate gene expression a case report 98. Combination therapy of tobramycin with in the emergence of antimicrobial resistance, which
and behaviour according dgalactose and lfucose to inhibit the virulence factors often leads to treatment failure and reduces the range of
to the local density of their
population.
PAIl and PAIIl cured an 18monthold infant with therapeutic options. So, there is an urgent need for effi
systemic and pulmonary infections, as determined by cient, costeffective and safe nonantibiotic therapy to
Entropy microbiological testing. prevent and treat UTIs without facilitating antimicrobial
A quantitative measurement Screening with the glycan arrays of the Consortium resistance. Inhibition of type 1 fimbriaemediated bacte
of the disorder in a system.
for Functional Glycomics revealed that the lewis a rial attachment to the bladder epithelium is a promising
The change in heat divided
by the absolute temperature trisaccharide, Galβ(13)[Fucα(14)]GlcNAc, is a high approach to achieve this goal109. Studies showed that
is the entropy change or cost affinity ligand for PAIIl99, with a dissocation constant αmannosides are the primary bladder cell ligands for
of the thermodynamic process. of 210 nM100. To reduce the complexity of the trisaccha UPEC and that the attachment event requires the highly
ride antagonists, glycomimetics based on the Fucα(14) conserved FimH lectins, which are located at the tip
Aglycon
The non-sugar component that
GlcNAc disaccharide — for example, the antagonist com of the bacterial fimbriae. A structure–function analysis
remains after hydrolysis of a pound 12 — were synthesized. by titration calorimetry showed that the residues of the FimH mannose binding
glycoside. experiments, increased entropy costs upon binding were pocket are invariant across 200 UPEC strains110.
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Table 2 | Carbohydrate epitopes used by bacteria and viruses for recognition and entry
Pathogen Binding epitope refs
Bacteria
Escherichia coli type 1 pili Manα(1-3)Manα(1-6)Man 109
Helicobacter pylori Neu5Acα(2-3)Galβ(1-4)Glc 109
Pseudomonas aeruginosa (PA-IL and PA-IIL) α-Galactoside and Lewis x 109
Neisseria gonorrhoea Galβ(1-4)GlcNAc 109
E. coli K99 Neu5Gcα(2-3)Galβ(1-4)Glc 109
E. coli CFA1 Neu5Acα(2-8)Neu5Ac 109
Klebsiella pneumonite Man 109
Campylobacter jejuni Fucα(1-2)Galβ(1-4)GlcNAc 109
E. coli K1 GlcNAcβ(1-4)GlcNAc 109
E. coli P Galα(1-4)Gal 109
E. coli S Neu5Acα(2-3)Galβ(1-4)GalNAc 109
Haemophylus influenzae Neu5Acα(2-3)Galβ(1-4)GlcNAc 109
Neisseria meningitidis Neu5Acα(2-3)Galβ(1-4)GlcNAc 109
Salmonella typhimurium Man 109
Streptococcus pneumoniae Neu5Acα(2-3)Gal 109
Streptococcus suis Galα(1-4)Galα(1-4)Glc 109
Viruses
Influenza A α(2-3)- or α(2-6)-linked sialic acid 186
Human parainfluenza virus type 1 α(2-3)SLN and sialyl Lewis x 187
Norwalk virus H and Le blood types
b
188
Rotavirus Sialylated glycans 189
Herpes simplex virus type 1 3-O-sulphated heparin sulphate 190
Calcivirus Blood group antigens 191
Corona virus Sialylated glycans 192
Murid herpes virus Glycosaminoglycans 193
Coxsackivirus A24 Sialylated glycans 194
Papilloma virus L1 Heparan sulphate 195
Polyomaviruses (JCV and BKV) α(2-3)- or α(2-6)-linked sialic acid 196
Simian virus SV40 GM1 ganglioside 197
Newcastle disease virus Sialylated glycans 198
BKV, B. K. virus; JCV, John Cunningham virus; Leb, Lewisb antigen; PA-IIL, fucose-binding lectin PA-IIL; PA-IL, PA-I galactophilic
lectin; SLN, sialyl lactoseamine.
More than two decades ago, various oligomannosides111 compound, methyl αdmannoside (compound 17)
and aromatic αmannosides112 that antagonize type 1 binds in the millimolar range118, but the most potent
fimbriaemediated bacterial adhesion were identified. Two monovalent antagonist reported so far, compound 22,
approaches have been taken to improve their affinity: binds with nanomolar affinity 117.
the rational design of ligands guided by information The reported affinities can be explained on the basis
obtained from the crystal structure of FimH, and the of the structure of the CRD that is located on the tip of
multivalent presentation of the αmannoside epitope. the FimH protein (FIG. 5). First, the hydroxyl groups at
The crystal structure of the FimH receptorbinding the 2, 3, 4 and 6 positions of mannose form an extended
domain was solved in 1999 (REF. 113) and the corre hydrogen bond network114,118. Second, the entrance to
sponding complex with oligomannoside3 (REF. 114) has the binding site formed by two tyrosines and one isoleu
recently become available. Despite this detailed know cine — the socalled ‘tyrosine gate’ — supports hydro
ledge of the binding event, few attempts to translate phobic contacts118. The aromatic aglycons of antagonists
π–π interaction
A non-covalent interaction
this information into lowmolecularmass antagonists — as occur in compounds 20 and 21, for example — can
between organic compounds have been reported112,115–117. A selection of monovalent establish energetically favourable π–π interactions with
that contain aromatic moieties. FimH antagonists is depicted in FIG. 5. The reference this tyrosine gate, leading to substantially improved
nATURE REvIEWS | Drug Discovery ADvAnCE OnlInE PUblICATIOn | 669
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a b 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 receptor–
ligand 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
stress119. 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
c (also known as the Tamm–Horsfall urinary glycopro
HO tein), which binds to FimH and is thought to function
OH HO O N
OH as a body defence against E. coli infections120. On the
OH OH
O OH O OH O O basis of simulations121, it is thought that forceinduced
HO OH
O O
N N O separation of FimH from its mannose ligand causes a
O O O OH
HO
O HO N COOCH3
OH conformational change of the binding pocket from
AcHN OH HO
HO AcHN a lowaffinity to a highaffinity conformation. Instead
Lewisa 12 13 of the application of competitive antagonists, allosteric
AcHN antagonists that are capable of stabilizing the lowaffinity
HO N
OH
N N OH conformation might lead to a successful therapy.
OH O O
O OH Although monovalent and oligovalent antagonists
N N O
O
4 O HO O with nanomolar affinity have been reported, there are
O O HO
HO N
HO
no data available regarding their pharmacokinetic prop
NHAc
14
OH erties. However, for the treatment of UTI, oral bioavail
HO
R ability and fast renal excretion to reach the targets in the
OH
O O OH urinary tract are prerequisites for therapeutic success.
O R O N N
N HO Rational design: challenges and lessons learned
O OH As in other fields that have spawned successful new
O O OH
O
R= NH
therapeutics (for example, monoclonal antibodies), years
O
O N N of effort have been required to understand the unique
NH N
challenges that are inherently linked to carbohydrate
HO derived drugs and to develop the basic skills and the
15 N N OH
N O
O specific knowledge to move from the excitement of
OH scientific discovery to the development of a new class
NH HO
O
O of therapeutics.
O Although animal lectins usually show a high degree
O HO of specificity for glycan structures, their singlesite
N N OH
R1 = NH
1
O
N O
O binding affinities are typically low. In biological systems,
O R
HO
OH functional affinity is often attained by the oligovalent
OH presentation of CRDs, either in an oligomeric protein
R1
HO
O O
O
(for example, cholera toxin122) or through clustering at
O O
HO OH cell surfaces (for example, asialoglycoprotein recep
O HO O
OH tor123). Additionally, the pharmacokinetic properties
OH
HO
HO
OH of carbohydrate hits, such as bioavailability or plasma
OH
16
halflife, are typically unsatisfactory for therapeutic
applications. Finally, although tremendously improved
Figure 4 | PA-iL and PA-iiL inhibitors. a | Binding sites of PA-I galactophilic lectin novel glycosylation protocols 124 and solidphase
(PA-IL) complexed with d-galactose (Protein Data Bank code: Nature1OKO
Reviews(REF. 170)
| Drug ).
Discovery approaches125 have become available, oligosaccharides
b | Binding sites of fucose-binding lectin PA-IIL complexed with l-fucose (PDB code:
are still only manufactured by cumbersome multistep
1GZT143). In parts a and b, the protein backbones are depicted in ribbon style,
carbohydrates are shown in ball and stick style and the grey spheres are Ca2+. syntheses.
c | The monovalent ligands compound 12 (REF. 101) and compound 13 (REF. 99) exhibit Therefore, the challenge is to mimic the structural
affinity for PA-IL and PA-IIL that is similar to that of Lewisa (REF. 100); the most potent information of a functional carbohydrate with a com
oligovalent ligand is compound 14 (REF. 102), but it has only a modest effect on pound that has druglike characteristics. The first step in
a per saccharide basis; the heterobifunctional glycodendrimer compound 15 this process is to understand the SAR of a carbohydrate
(REF. 104) and the low-molecular-mass glycomimetic compound 16 (REF. 105) lead, specifically the contribution made by each func
bind to both PA-IL and PA-IIL from Pseudomonas aeruginosa. tional group to binding as well as the threedimensional
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© 2009 Macmillan Publishers Limited. All rights reservedREVIEWS
presentation of the pharmacophores. based on this pharmacokinetic properties — in particular, improved
information, it is possible to identify glycomimetics bioavailability and serum halflife — while minimizing
that are preorganized in their bioactive conformation toxicity and cost of synthesis. In the past, the develop
— that is, which will adopt their bound conformation in ment of carbohydratederived drugs was often not
solution. In addition, the mimics should show improved entirely focused on simultaneously solving all of the
HO HO HO OH
OH OH
HO O HO O HO O
HO HO HO
OCH3 O O
17 (REF. 118) 18 (REF. 115) 19 (REF. 116)
RIP = 1 RIP = 85 NO2 RIP = 440
HO OH HO OH HO OH
HO O HO O HO O
HO HO Cl HO
O O O
O O O O
NO2 Cl N
H O
20 (REF. 116) 21 (REF. 112) 22 (REF. 117)
RIP = 110 RIP = 717 RIP = 6900
HO OH
OH
O OH
OH
HO O
O OH
HO O
HO
HO O (H2C)3 HOOH O O
OH
O O
OH OH OH
(H2C)2 OH N N OH
HO HO (H2C)3 N N
O HO OH N N
OH O OH OH
O HO O O
HO O (H2C)2 OH HO O OH
O O O N N
O O
O (CH ) (H2C)3 O
HOHO N N
O (CH2)3
2 2 O O S S N N N N N
O N
(CH2)2 OH
O (CH2)2 HO OH
O N N
N N N N OH
O H H O
HO HO O (CH2)3 OH
HO O
H H O O O OH
OH N N N N OH
HO HO
HO 25 (REF. 173)
HO OH 23 (REF. 171) O S S O HOOH OH
OH OH RIP = 80 HO
RIP = 130
HO OH OH
OH HOHO
24 (REF. 172)
RIP = 780 O OH
O
HO HO O O
OH
OH OCH3
O
HO OH HO
HO
O
O OMe
OH
NHOH
x y O O
O O OH HO O
HN OO NH O HO OH N
HO O NH H
O OH HO OCH3
HO
HO OH O
NO2
HOOH O O O
HO HO O OH
26 (REF. 175) 27 (REF. 116) 28 (REF. 170)
RIP = 30–39 RIP = 4900 RIP = 330
OH
HO OH HO
Figure 5 | FimH antagonists. The crystal structure representation shows the mannose derivative compound 21 docked
to the mannose-binding pocket of FimH (Protein Data Bank code: 1KFL). The relative inhibitory potencies
Nature Reviews (RIPs)
| Drug of the
Discovery
FimH antagonists compounds 18 to 28 are based on methyl α-d-mannoside (compound 17; RIP = 1). 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.
nATURE REvIEWS | Drug Discovery ADvAnCE OnlInE PUblICATIOn | 671
© 2009 Macmillan Publishers Limited. All rights reservedREVIEWS
above requirements and some highprofile failures reducing the entropy costs on binding. For Eselectin,
resulted, notably in the field of selectin antagonists. this strategy was successful (see complex 30 in FIG. 6a).
nevertheless, rationally designed glycomimetics have As elucidated by Xray 126 or STD nMR134 studies, the
the potential to reap the rewards of a relatively untapped GlcNAc moiety does not interact with the binding site
source of novel therapeutics for wideranging and and serves solely as a linker that positions the galactose
important biological and medical applications. and the fucose moiety in the correct spatial orientation.
It was successfully replaced by noncarbohydrate link
Understanding native interactions. The starting point ers141,142. In addition, steric repulsion deriving from
for the rational design of glycomimetics is the analysis properly placed substituents on the linker moiety can
of the binding characteristics of the carbohydrate–CRD further improve the preorganization of the core and, as
binary complex. The threedimensional structure of a result, the affinity of the corresponding antagonist 130.
the lectin or the carbohydrate–lectin complex has been Furthermore, the preorganization of the carboxylate was
solved for a number of therapeutically interesting targets. optimized as well, revealing (S)cyclohexyl lactic acid as
Thus, E, P and lselectin cocrystallized with slex or the best mimic of neu5Ac141.
PSGl1 (Pselectin glycoprotein ligand 1)126, siaload If the target lectin offers a wellstructured binding
hesin cocrystallized with 3′sialyl lactose127, or DCSIGn pocket, the free energy of binding can be improved
cocrystallized with the pentasaccharide GlcNAc2Man3 by incorporating additional enthalpic contributions.
(REF. 128) hold valuable information for the rational design Successful examples are the neuraminidase inhibitors
of glycomimetics. In cases in which the structure has not zanamivir7 and oseltamivir 9. For the influenza viral coat
yet been solved, homology models can be generated — as protein neuraminidase, the natural substrate neu5Ac
is the case for MAG, for example129. and the corresponding glycal neu5Ac2en (compound
Detailed insight into the binding event can be gained 31), which mimics the transition state of the hydrolytic
by nuclear magnetic resonance (nMR) experiments. For reaction, have only millimolar to micromolar affinities.
example, the bound conformation of a functional carbo The improved affinities of the transition state analogues
hydrate ligand in the CRD of the target lectin can be deter zanamivir and oseltamivir result from a guanidinium
mined using transferred nuclear Overhauser effect (nOE)130. substitution in the 4 position, enabling the forma
In addition, the binding epitope can be identified by satu tion of a new salt bridge7, or from the replacement of
ration transfer difference nMR spectroscopy (STD NMR the glycerol side chain in the 6 position, leading to a
spectroscopy)131. This technique has been used to study new, favourable lipophilic interaction by induced fit 9
interactions of carbohydrate ligands with the rotavirus (FIG. 6b).
receptor, vP8 (REF. 132), the anticarbohydrate tumour Finally, multivalency frequently occurs in nature
associated antibody GSlA1 (REF. 133), Eselectin134 and and leads to tight binding in situations in which univa
MAG87,135. Overall, transfer nOE nMR and STD nMR lent protein–ligand binding is weak143–145. Recognition
experiments allow a rapid insight into the binding char of carbohydrate ligands by bacterial and mammalian
acteristics of carbohydrate–lectin interactions and can lectins are examples of this phenomenon. For the spe
replace, at least partially, Xray investigations and the cific inhibition of these recognition events, oligovalent
timeconsuming mapping of binding epitopes by chemical ligands have been proposed (see, for example, FIGS 4,5).
means136. However, the design of tightbinding oligovalent ligands
is, for the most part, an empirical endeavour. Tailored
Enhancing binding affinity. The generally low affinity oligovalency, whereby the spacing of a limited number
of carbohydrate–lectin interactions is a consequence of of tethered branches is matched to that between adja
shallow binding sites of lectins, leading to a high solvent cent sugar binding sites of a protein or a protein cluster,
accessibility of the complex forming hydrogen bonds potentially offers substantial increases in avidity for the
and salt bridges. Owing to large offrates (koff ), the target143,146,147.
binary complexes are characterized by short dissociative
Nuclear Overhauser effect
The cross-relaxation between halflives (t1/2), typically in the range of seconds — as Pharmacokinetics. Unfortunately, only limited phar
two nuclei, which is observable shown for selectins and their physiological ligands137–139, macokinetic data are reported for any carbohydrate or
through the longitudinal the carbohydraterecognizing antibody GSlA1, slea glycomimetic. For oral absorption by passive permeation
magnetization of a given (REF. 133) and MAG antagonists135. Given that, for a thera through the membrane barrier of the small intestine148,
nucleus after a second nucleus
is perturbed from equilibrium.
peutic application, the t1/2 of a drug–target binary com there are limitations regarding molecular mass, polarity
plex is expected to be in the range of minutes to a few and the number of hydrogen bridge donors and accep
STD NMR spectroscopy hours, improving the koff of glycomimetic compounds is tors149. The hydrophilic nature of oligosaccharides
(Saturation transfer difference mandatory for therapeutic applications140. caused by the large number of hydroxyl groups and
nuclear magnetic resonance
Often, mammalian lectins undergo numerous charges (sulphates and carboxylates) makes their oral
spectroscopy). An experiment
in which spin-diffusion of the directed, but weak, interactions with their ligands. A availability virtually impossible. Therefore, when glyco
nuclear Overhauser effect specific example, the interaction of slex with Eselectin, mimetics are designed, the pharmacokinetic as well
spreads magnetization is outlined in FIG. 6a. It consists of six solventexposed as the pharmacodynamic profile should be adjusted.
throughout the hydrogen hydrogen bridges and a salt bridge (to produce complex Possible strategies to improve passive absorption are the
nuclei of a receptor, which is
partially transferred to the
29). One possible approach to improve affinity is to pre bioisoteric replacement of crucial groups150 or a pro
hydrogen nuclei of a binding organize the antagonist in its bioactive conformation drug approach151. A successful example of the prodrug
ligand. to compensate for the low enthalpic contributions by approach is oseltamivir, which is an ester prodrug. Once
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