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5
Antiadhesive
Jonathan Cramer
1
University of Basel, Department of Pharmaceutical Sciences, Pharmacenter, Klingelbergstrasse 50, Basel
CH-4056, Switzerland
2
Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1,
Düsseldorf DE-40225, Germany
3
WuXi AppTec UK LTD, 5 New Street Square, London EC4A 3TW, United Kingdom
Carbohydrates and Glycomimetics
1,2
, Lijuan Pang3, and Beat Ernst
1
5.1 Introduction
Infectious diseases are still a major cause of death, disability, and social and economic
disorder for millions of people throughout the world. Poverty, poor access to health
care, human migration, emerging disease agents, and antibiotic resistance all contribute to the expanding impact of these illnesses[1]. Prevention and treatment strategies
for infectious diseases are derived from a thorough understanding of the complex
interactions between specific viral or bacterial pathogens and the human host.
Glycans are found on the surfaces of all bacteria and viruses, as well as on their
hosts. Thus, a majority of interactions between microbial pathogens and their hosts
are based on the interaction of carbohydrate epitopes on the one hand and glycanbinding receptors on the other hand[2]. This initially leads to the colonization of
host epithelial surfaces, a prerequisite for spreading infection. In this chapter, the
status of the development stage of Antiadhesive Carbohydrates and Glycomimetics is
presented by means of some selected examples.
5.1.1 Carbohydrate–Protein Interactions inViral Adhesion
toHost Cells
The surfaces of viruses and host cells are densely covered with diverse glycans. Many
viruses exploit carbohydrate–protein interactions for adhesion to host cells, initiation of
virus internalization, and evasion of immune surveillance[3–5]. Whereas bacterial adhesion is most commonly mediated by the interaction of bacterial lectins with host-derived
glycoproteins, viruses have a more diverse arsenal of adhesion mechanisms. Similar to
bacteria, some viral pathogens display carbohydrate-binding proteins on their surface
that can specifically recognize certain host glycans [5]. A well-studied example is the
interaction of the hemagglutinin glycoprotein expressed on the surface of influenza
Carbohydrate-Based Therapeutics, First Edition. Edited by Roberto Adamo and Luigi Lay.
© 2024 WILEY-VCH GmbH. Published 2024 by WILEY-VCH GmbH.
5 Antiadhesive Carbohydrates and Glycomimetics
132
A viruses with sialic acid-containing host glycans. Furthermore, glycosaminoglycans,
acidic linear polysaccharides that commonly decorate host cell surfaces, can serve as an
initial attachment factor for many different viruses. Alternatively, endocytic carbohydratebinding receptors on host cells can promote adhesion to viral envelope glycoproteins and
subsequent internalization[3, 4]. An archetypical example of this process, the interaction
of the myeloid C-type lectin DC-SIGN with viral glycoproteins, will be discussed in this
book chapter. Many viruses employ different strategies for adhesion simultaneously.
Besides mediating the interaction with sialic acid receptors, the influenza A hemagglutinin glycoprotein is also heavily modified with N-linked glycans. These structures are recognized by DC-SIGN and mediate a secondary, sialic acid-independent adhesion and
entry mechanism into host cells[6].
5.1.2 Bacterial Adhesins and Antiadhesion Therapy
An essential step of bacterial infection and pathogenesis is the adherence of bacteria
to cell surfaces of the host tissue, granting the bacteria substantial resistance to natural defense mechanisms, mechanical shear stress, and antibiotics[7]. Bacteria can
express more than one type of adherence factors or “adhesins.” Most of these adhesins are lectins that bind directly to cell-surface carbohydrate motifs on glycoproteins
or glycosphingolipids via carbohydrate-recognition domains (CRDs)[8, 9]. Bacterial
lectins commonly exist in the form of elongated, hair-like, multi-subunit protein
appendages, known as fimbriae (hair) or pili (threads), protruding from the surface
of bacteria[7, 8]. Although carbohydrate–lectin interactions are generally of low
affinity, such pili structures provide a multivalent, Velcro-like binding to epithelial
surfaces, hence facilitating bacterial survival and invasion[8, 10]. Therefore, antiadhesive agents that block bacterial adherence to host tissues may offer a novel strategy to combat infectious diseases.
Similar to animal lectins, bacterial lectins bind to terminal sugar residues or internal glycan sequences present in linear or branched oligosaccharide chains [11].
Since Sharon etal. first described bacterial surface lectins in 1970s,[12] researchers
have identified a large fraction of the carbohydrate epitopes (“adhesin receptors”)
used by bacteria for colonization and entry into host tissues (Table 5.1) [31].
Although the natural carbohydrate epitopes show effectiveness in blocking microbial adhesion, their susceptibility to enzymatic degradation and undesirable pharmacokinetic properties hamper their clinical applications [32]. Based on resolved
protein structures, structure-based rational design advanced the identification and
optimization of antiadhesive glycomimetics, allowing improved metabolic stability,
binding selectivity, and bioavailability[33–35]. In the era of increasing antimicrobial resistance, one exceptional advantage of antiadhesive therapeutics is that they
do not kill or restrict the growth of the pathogens and are therefore less likely to
promote antibacterial resistance. Additionally, antiadhesive agents could potentially reduce overuse of broad-spectrum antibiotics and thus prevent long-lasting
detrimental effects on the healthy human microbiota. Furthermore, target-specific
antiadhesion therapy makes precision antimicrobial treatment possible[36, 37].
The search for FimH and PA-IL/IIL inhibitors depicts representative examples of
modern antiadhesive therapeutics.
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
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5.1.3 Selected Examples
In this chapter on Antiadhesive Carbohydrates and Glycomimetics, we are forced to
limit ourselves to a few selected examples. From a plethora of therapeutic targets for
which the involved carbohydrate ligands and lectins have been elucidated, only the
most prominent examples, namely DC-SIGN and the virulence factors FimH, PA-IL,
and PA-IIL, were selected for discussion.
The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed
on macrophages and dendritic cells (DCs). It has been identified as a promiscuous
entry receptor for many pathogenic agents, including pandemic viruses such as
SARS-CoV-2, ebola, and HIV[38]. The virulence factors FimH, PapG, PA-IL, and
PA-IIL are expressed by pathogenic bacteria that represent an immediate or future
threat to public health in the light of emerging antibiotic resistance.
FimH is one of the most studied adhesins expressed by uropathogenic Escherichia
coli (UPEC) strains because it is a key determinant of urovirulence [39]. Urinary
tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs) are
becoming increasingly important threats to human health, and the antiadhesive
strategy emerged as a relevant alternative therapeutic approach. Recently, the FimH
antagonist GSK3882347 entered Phase I clinical trials in a collaboration between
Fimbrion Therapeutics and GlaxoSmithKline.
Finally, Pseudomonas aeruginosa produces biofilms that can cause chronic opportunistic infections, which often cannot be treated effectively with traditional antibiotics [40]. Since P. aeruginosa is considered a model organism for the study of
antibiotic-resistant bacteria, its virulence factors PA-IL and PA-IIL were extensively
studied.
133
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into
Myeloid Cells
5.2.1 Introduction
C-type lectin receptors (CLRs), a class of proteins expressed on the membrane of
myeloid cells such as DCs and macrophages, are often exploited as entry receptors
by viral pathogens[38, 41]. Physiologically, CLRs recognize conserved carbohydrate
epitopes on diverse pathogens and initiate tailored immune responses. However,
some viruses have developed the ability to circumvent the physiological function of
CLRs and infect myeloid cells themselves (cis-infection) or other cells under mediation of myeloid CLRs (trans-infections). The CLR DC-specific ICAM-3-grabbing
nonintegrin (DC-SIGN, CD209) has been proven vulnerable to viral exploitation,
adhesion and internalization of virus particles can result in trafficking to nonlysosomal compartments and virus persistence in a protected intracellular environment.
Besides this, DC-SIGN has been demonstrated to serve as a receptor for many other
viruses, such as dengue, zika, ebola, and coronaviruses [38]. Because of their
5 Antiadhesive Carbohydrates and Glycomimetics
134
Table5.1 Carbohydrate epitopes used by bacteria for colonization and entry in host
tissues.
Pathogen Adhesin Binding epitope References
Campylobacter
jejuni
Escherichia coli Type-1 fimbriae Manα(1-3)Manα(1-6)Man [14]
Haemophilus
influenzae
Helicobacter pylori BabA Lewis B [21]
Klebsiella
pneumoniae
Mycobacterium
tuberculosis
Neisseria
gonorrhoeae
Pseudomonas
aeruginosa
Salmonella
typhimurium
Streptococcus
pneumoniae
Streptococcus suis SadP Galα(1-4)Galα(1-4)Glc [30]
Flagella, LPS Fucα(1-2)Galβ(1-4)GlcNAc [13]
P fimbriae Galα(1-4)Gal [15]
S fimbriae Neu5Acα(2-3)Galβ(1-4)
K99 fimbriae Gangliosides GM3,
CFA1 AsialoGM1, Lewis A [18, 19]
HMW1 adhesin Neu5Acα(2-3)Galβ(1-4)
SabA Sialyl Lewis X [22]
Type-1 fimbriae Man [23]
Heparin-binding
hemag-glutinin adhesin
(HBHA)
Opa proteins LacCer, Neu5Acα(2-3)
PA-IL (LecA) Galactosides [26]
PA-IIL (LecB) Lewis A, Fuc [27]
Type-1 fimbriae Man [28]
Carbohydrate-binding
modules of
β-galactosidase, BgaA
GalNAc
Neu5Glcα(2-3)Galβ(1-4)Glc
GlcNAc
Heparan sulfate [24]
Galβ(1-4)GlcNAc, syndecans,
heparan sultate
Lactose, N-acetyllactosamine, Neu5Acα(2-3)
Gal
[16]
[17]
[20]
[25]
[29]
potential to interfere with viral adhesion, carbohydrate-based molecules and glycomimetic drugs targeting DC-SIGN are of tremendous interest. This treatment strategy circumvents common resistance mechanisms through attenuation of virulence
and provides a host-directed pharmacological response to not only established but
also newly emerging infections with pandemic potential.
DC-SIGN is anchored to the cell membrane by a hydrophobic neck domain that
induces tetramerization (Figure5.1a). Carbohydrate ligands are bound to the CRD
by a calcium ion acting as a cofactor in the primary binding site (Figure 5.1b).
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
(a) (b)
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Figure5.1 Structure of DC-SIGN. (a) Schematic depiction of DC-SIGN domain organization.
(b) The carbohydrate-binding site of DC-SIGN in complex with α-1,2-mannobiose (PDB
2IT6). Secondary interaction sites commonly targeted by glycomimetics are highlighted.
DC-SIGN naturally binds to underprocessed high-mannose-type glycans (e.g.
GlcNAc2) that are abundantly presented on viral envelope glycoproteins.
Man
9
Fragments of this glycan as well as mannose itself also bind to DC-SIGN, albeit with
lower binding affinity. In addition, DC-SIGN recognizes fucosylated glycans such as
Lewis-type and ABO antigens. Canonically, mannose or fucose epitopes coordinate
with the central calcium ion via their 3-OH and 4-OH groups[44, 45]. However, it
has been demonstrated that mannose ligands are able to bind in a variety of transient binding modes, also employing other hydroxyl functions [46]. Various concepts have been employed to identify glycomimetic ligands that utilize secondary
binding sites to achieve higher monovalent affinity toward DC-SIGN. Besides
ligand-/structure-based design[47, 48], combinatorial[49, 50] and fragment-based
approaches[51, 52] were successful. Important sites for additional secondary interactions in an extended binding site are highlighted in Figure5.1: In the long loop,
Val351mediates binding of Lewis-type antigens to DC-SIGN[45, 53]. A hydrophobic subsite in the vicinity of Phe313 is an additional target for glycomimetics[52,
54]. This allosteric pocket, as well as several other distal areas, have been identified
as binding sites for noncarbohydrate fragments. Finally, Glu358 and Ser360have
been shown to act as binding partners for positively charged residues[47]. In general, druggability of DC-SIGN and other CLRs has been soundly demonstrated by
experimental and computational approaches[51].
Approaches for the development of carbohydrate-based inhibitors of viral attachment and entry can be categorized into two different groups. Firstly, natural monoor oligosaccharide ligands of DC-SIGN have been utilized for the synthesis of
various multivalent systems. A second approach relies on the design of carbohydrate derivatives or glycomimetics that surpass the affinity of natural ligands and
can be employed as monovalent therapeutics or utilized for the construction of multivalent systems with improved affinity.
135
5 Antiadhesive Carbohydrates and Glycomimetics
136
5.2.2 DC-SIGN Ligands Employing Natural Carbohydrate Epitopes
Section5.2.2. gives an account of the multivalent scaffolds employed for the presentation of mannose- and fucose-based epitopes (Table5.2), as well as general trends
observed in several studies. It must be emphasized that the multitude of different
assay formats used across the literature employing either recombinant protein or a
cellular system renders a direct comparison of multivalent ligands impossible. In
any case, numeric affinity values are mainly determined by specific assay setups and
are difficult to compare. This problem could easily be avoided by including monovalent controls (e.g. methyl α--mannoside) in every study and reporting relative
affinities compared to this control. Unfortunately, this easily implementable solution is only applied sporadically, and we would like to encourage this practice for
future publications of affinity data for multivalent ligands in general.
Table5.2 Overview over multivalent systems employed for DC-SIGN targeting.
Multivalent scaffold Carbohydrate epitopes References
Dendrimers Monosaccharides [55, 56]
α-1,2-mannobiose [57]
Glycan-derived
oligosaccharides
[58, 59]
Nanoparticles Monosaccharides [60–62]
α-1,2-mannobiose [61–63]
Glycan-derived
oligosaccharides
Polymers Mannose [66–69]
Other scaffolds Monosaccharides [70–73]
[64, 65]
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
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5.2.2.1 Dendrimers
Hyperbranched dendritic polymers and polyamido amine (PAMAM) dendrimers
TM
(Boltorn
) have been extensively used for the construction of multivalent DC-SIGN
ligands [55–57]. As a general trend observed in these studies, only larger, highvalency compounds showed binding to DC-SIGN[55]. The most potent derivatives
blocked DC-SIGN/gp120interaction with a nanomolar IC
6
resenting a 10
-fold affinity enhancement compared with the monovalent mannose
in an ELISA assay, rep-
50
control[56]. It was also found that higher generation dendrimers, which were characterized by a larger particle size and wider glycan spacing, were more potent
DC-SIGN binders[57]. Importantly, DC-SIGN binding and uptake of the dendrimers into DCs did not result in activation or maturation. In an impressive application,
up to 360 α-1,2-mannobiose epitopes were displayed on [60]fullerene-based dendrimer scaffolds[58]. These particles were shown to inhibit the DC-SIGN-mediated
+
infection of Jurkat DC-SIGN
cells with dengue and zika virus pseudotypes in pico-
molar concentration.
5.2.2.2 Nanoparticles
Gold nanoparticles (GNPs) decorated with linear and branched mannose oligosaccharides have been shown to inhibit the infection of human T cells in an HIV trans-
infection assay in nano- to subnanomolar concentrations [59, 60]. Similarly,
mannose- or α-1,2-mannobiose-functionalized GNPs inhibited ebola glycoprotein-
+
driven infection of DC-SIGN
cells with up to 100 pM affinity[61]. Interestingly,
glycan density on the GNPs, rather than the exact composition of the oligomannose
ligand, determined the outcome of the experiment. Again, treatment of DCs with
functionalized GNPs did not lead to maturation or induction of DC-SIGN-associated
signaling, thus highlighting the pure targeting function of glyconanoparticles[62].
CdSe/ZnS quantum dots (QDs) functionalized with 369 carbohydrate epitopes
potently bound to soluble recombinant DC-SIGN with an apparent K
6
compared with monovalent ana-
increase in binding affinity by a factor of 1.5
× 10
of 0.6 nM, an
D
logs[63, 64]. Linker length was found to be a critical factor for the affinity of functionalized QDs. Long, more flexible PEG11 linkers resulted in weaker binding
affinity compared with shorter PEG3linkers. The authors hypothesized that the
longer linker chains suffered from a more pronounced entropic penalty upon ligand
binding, ultimately diminishing the overall free energy of binding. In an EBOVgppseudovirus infection assay, the QDs proved to be highly potent blockers of
DC-SIGN-mediated infection, inhibiting virus internalization with an IC
as low as
50
0.7 nM. Despite the high in vitro activity of CdSe/ZnS QDs, the authors acknowledge the severe drawbacks of the inherently cytotoxic nanoparticles for biological
applications.
A notable feature of small, spherical nanoparticles is their efficient discrimination between DC-SIGN and the related receptor DC-SIGNR[61]. Despite an identical protein fold and high sequence similarity, these two proteins differ in the
structural organization of their CRDs in their respective tetrameric assembly.
137
5 Antiadhesive Carbohydrates and Glycomimetics
138
Whereas DC-SIGN aligns its CRDs vertically, orthogonal to the cell membrane
(“closed flower” arrangement), DC-SIGNR orients its CRDs perpendicular to the
plane of the cell membrane, thereby pointing each CRD in a different direction
(“open flower” arrangement). As a result, DC-SIGN binds globular ligands with
four CRDs simultaneously, capitalizing on additional multivalency effects and
resulting in increased binding affinity. A single DC-SIGNR tetramer, however, binds
globular ligands with a single CRD and, as a result, has lower affinity.
5.2.2.3 Polymers
Linear polymers, as well as nonlinear brush and bottlebrush polymers presenting
mannose epitopes, have also been explored for their application toward DC-SIGN
binding [65–67]. It was found that high valency was the main determinant for
DC-SIGN affinity, whereas, unlike for other studied lectins, the degree of branching
did not impact binding affinity significantly. When the polymer dextran was functionalized with mannose or n-heptyl α--mannoside (HM)[68], the polymer with
the highest valency (902 HM epitopes) showed the highest binding affinity with an
value in the subnanomolar range. This represents a 33-fold affinity enhance-
IC
50
ment per HM epitope compared with the monovalent control. The high affinity was
corroborated in a trans-infection assay, where DC-SIGN-mediated trans-infection of
permissive fibroblasts was inhibited with an IC
of 20 pM, which is a 104-fold
50
enhancement compared to the monovalent control methyl α--mannoside.
Polymers based on a poly--lysine backbone can successfully inhibit viral adhesion
to DC-SIGN-expressing cells with sub-nanomolar affinity[69]. It was shown that
long polymers forming larger particles by aggregation were more potent inhibitors
of glycoprotein binding compared to shorter, fully soluble analogs. A notable feature of poly--lysines is the fact that they are nonimmunogenic, biocompatible, and
efficiently degraded by target cells, thus highlighting their potential for biological
applications[69–72].
5.2.2.4 Other Multivalent Scaffolds
Besides the common multivalent carriers detailed above, a number of other systems
have been used for targeting DC-SIGN. The most potent compounds from a study
with calixarene and thiacalixarene scaffolds were able to inhibit cis-infection of
+
DC-SIGN
cells in nanomolar concentrations [73, 74]. In another approach,
dynamic micelles based on mono- and trivalent mannoside glycolipids have been
investigated[75]. Finally, nanocarbon-based scaffolds carrying mannose dendrimers have been investigated for their ability to inhibit viral attachment to DC-SIGNexpressing cells[76]. In these experiments, single- and multiwall carbon nanotubes
and nanohorns prevented DC-SIGN-mediated ebola virus infection at low concen-
1
trations down to 0.37 μg ml
.
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
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5.2.3 DC-SIGN Ligands Employing Carbohydrate Derivatives
or Glycomimetics
Besides improving the affinity of weak ligands by their multivalent presentation,
the affinity of individual ligands can be enhanced by structural modifications of
natural carbohydrates, enabling additional favorable interactions with the target.
Mitchell etal. reported DC-SIGN ligands based on C2-branched mannose derivatives obtained by a Kiliani ascension of fructose. In particular, 2-C-aminomethyl-mannose (1) stood out in a surface plasmon resonance (SPR) competition assay with
a 48-fold improved binding affinity for DC-SIGN compared with mannose (from
17.1 to 0.35
mM) [77]. Glycomimetic DC-SIGN ligands identified from a library
based on the shikimic acid core were reported by Garber etal.[49]. In a solid-phase
fluorescence assay, the most potent hit 2 displayed an IC
of 3.2 mM, which is about
50
a fourfold increase compared to the positive control N-acetylmannosamine. When
this molecule was subsequently incorporated into a linear polymer obtained by
ring-opening metathesis, a multivalent ligand with micromolar affinity
=2.9 μM) was obtained. In an attempt to target two hydrophobic pockets adja-
(IC
50
cent to Phe313, Tomašić etal. introduced large hydrophobic substituents on a flexible bifurcated glycerol linker in the C1-position of α--mannose [48]. The most
potent analog 3, bearing two naphthyl substituents, showed an IC
value of 40 μM
50
in a competitive solid-phase immunoassay. Whereas molecular docking studies
confirmed the intended binding mode targeting Phe313, molecular dynamics simulations starting from the predicted binding mode revealed residual flexibility of the
ligand, allowing additional interactions for the hydrophobic substituents
(Figure5.2).
The Bernardi group pioneered the use of cyclohexane-based pseudo-dimannoside
mimetics as DC-SIGN ligands. The glycomimetic scaffold 6 is accessible by oxidation of dimethyl (1S,2S)-cyclohex-4-ene-1,2-dicarboxylate (4) followed by selective
139
Figure5.2 Monovalent carbohydrate derivatives and glycomimetics as DC-SIGN ligands.
5 Antiadhesive Carbohydrates and Glycomimetics
140
Scheme 5.1 Synthesis of α-1,2-mannobiose mimetics. (a) MCPBA, rt; (b) Cu(OTf)2,
2-bromoethanol, rt; (c) NaN
, 50 °C; and (d) TMSOTf, –20 °C.
3
alkoholysis of the resulting epoxide 5[78]. This intermediate was then converted to
dimannoside mimetics of the general formula 9[79] (Scheme5.1).
Since the first report of these glycomimetics, their binding affinity and selectivity
toward the related CLR langerin, an anti-target in HIV therapy, have been continuously optimized. A key improvement was achieved by replacing the two carboxylic
esters by benzylamides (10)[54, 80]. This enabled extended hydrophobic contacts to
Val351 located in the long loop (see Figure 5.1), increasing potency to ca.
=300 μM in an SPR competition assay. The introduction of an amino substitu-
IC
50
ent in position 6 of the mannose moiety yielded fully selective DC-SIGN ligand 11
with virtually no affinity toward langerin[81]. By virtual screening of a fragment
library, an ammonium binding site was identified, which provided the incentive for
the synthesis of a triazol library at the axial 2-position of the mannose moiety[47].
The best ligand 12 displayed an IC
of 76 μM by SPR and a KD of 52 μM by ITC
50
(Figure5.3).
Figure5.3 Representative structures of pseudo-dimannoside glycomimetics and their
binding affinities to DC-SIGN.
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