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5 Antiadhesive Carbohydrates and Glycomimetics
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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
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
increase in binding affinity by a factor of 1.5 × 10
compared with monovalent ana-
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
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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
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
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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.
5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
Bernardi’s DC-SIGN ligands were designed for multivalent applications. Grafting
of pseudo-dimannoside 10 and a related pseudo-trisaccharide analog on a thirdgeneration Boltorn dendrimer scaffold yielded multivalent ligands showing strong
binding in an SPR competition experiment (IC
of DC-SIGN-mediated cis- (IC
=20 nM) and trans-infection (IC50=32–62 nM) in
50
=1–2 μM) and efficient inhibition
50
an Ebola virus infection model[82]. However, for high-valency derivatives, the multivalent presentation of glycomimetic 10 led to solubility issues[83]. When the same
ligand 10 was presented on rigid molecular rods[84], affinity tended to increase
with the length of the rod-like linker. The most potent compound, dubbed
Polyman26 (13), reached the lower detection limit (IC
SPR assay and efficiently prevented trans-infection of CD4
tion assay with an IC
of 24 nM. Treatment of human immature monocyte-derived
50
=<5 μM) in a competitive
50
+
T cells in an HIV infec-
DCs with 13 induced the activation of immune responses with an elevated production of the chemokines CCL3, CCL4, and CCL5, as well as proinflammatory
cytokines IL-1β, IL-6, and TNFα, highlighting the potential of DC-SIGN ligands for
immunomodulatory applications[85]. The same compound has been investigated
+
for its potential to block DC-SIGN-mediated trans-infection of ACE2
by SARS-CoV-2pseudovirions. With an IC
of 94 nM, the activity of 13 proved to be
50
Vero E6 cells
comparable to that in the HIV assay[86] (Figure5.4).
Another class of mannose-based DC-SIGN ligands has been disclosed recently[87].
Supported by molecular docking experiments, a focused library of triazole glycomimetics with modifications in positions 2 and 6was synthesized. Compounds with
an aromatic substitution in position 2were found to efficiently engage in a hydrophobic interaction with Val351. An extension of the anomeric position toward the
ammonium binding pocket then generated 14, which showed a K
of 31 μM in an
D
ITC assay. A multivalent poly--lysine polymer modified with a derivative of this
glycomimetic ligand inhibited the trans-infection of susceptible Vero E6 cells by a
SARS-CoV-2pseudovirus with an IC
of 4 nM (Figure5.5).
50
141
5.2.4 Conclusion and Perspectives
Adhesion of pathogens to DC-SIGN and subsequent internalization is a conserved
process that plays a role in a multitude of infectious diseases, predominantly of viral
origin. Inhibition of DC-SIGN-mediated viral attachment to the cell surface with
carbohydrate-derived therapeutics provides a host-directed pharmacological
response, offering an attractive strategy for the development of broad-spectrum
antivirals. Despite the affinity improvement from millimolar affinity for the monovalent natural ligand -mannose to low-micromolar monovalent mimetics, the
affinities are not yet sufficient to compete with the multivalent interaction between
DC-SIGN clusters on the cellular surface and arrays of viral envelope glycoproteins.
Thus far, only multivalent therapeutics have shown the potential for efficient competition in biologically relevant concentrations. Whereas multivalent compounds
employing natural carbohydrate epitopes can reach impressive potency down to
picomolar concentrations, an open question is the selectivity of these systems for
DC-SIGN over other lectins binding mannose, such as Langerin and other CLRs or
Figure5.4 Structure of Polyman26 (13).
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5.3 The Bacterial Adhesin FimH
Figure5.5 Structure of glycomimetic 14 and its multivalent presentation.
the mannose-binding lectin (MBL), which often also play a role in immune regulation. Here, the multivalent presentation of selective glycomimetics holds great
potential to yield highly potent and selective therapeutics.
5.3 The Bacterial Adhesin FimH
5.3.1 UTIs and FimH
UTIs affect millions of people and account for significant morbidity and high medical costs worldwide[88, 89]. Statistics show that about 50% of women experience a
UTI in their lives, and about 60% of them experience recurrent infections shortly
after the preceding treatment[90, 91]. UTIs are primarily caused by Gram-negative
UPEC, which constitute up to 90% of the diagnosed cases. UPEC infection involves
a well-defined multistep cascade that has been demonstrated in mouse cystitis models and human UTIs[92]. At first, UPEC adhere with their type 1 fimbriae (pili) to
mannosylated glycoprotein receptors, mainly uroplakin-Ia (UPIa), located on the
surface of the urinary bladder mucosa[93]. This adhesion event, a prerequisite for
bacterial invasion, prevents UPEC from being cleared by the shear stress of urine
flow and triggers bacterial invasion into urothelial cells. After entering the host
cells, UPEC starts replicating to form biofilm-like intracellular bacterial communities (IBC), protecting the bacteria from antibiotics treatment and host innate
immune responses. Virtually all the clinical UPEC isolates express pili, which are
uniformly distributed on their surface, amounting to 100–400 copies per cell[94–96].
Structurally, type 1 pili are 7 nm wide, several micrometers long, rod-like fibers.
Assembled by the chaperone/usher pathway, the pilus rod is a right-handed helical
structure composed of numerous immunoglobulin-like (Ig) FimA subunits, terminated by the fimbrial tip comprising FimF, FimG, and the lectin FimH.
143
5.3.2 FimH CRD
FimH (29 kDa) consists of two Ig-like domains: the N-terminal lectin domain
(FimH
domain (CRD), and the pilin domain (FimH
), which contains an α--mannose-specific carbohydrate-recognition
LD
), which connects FimH to the pilus
PD
rod and regulates the switch between the low- and high-affinity states of the lectin
5 Antiadhesive Carbohydrates and Glycomimetics
(a)
(b)
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144
Figure5.6 Schematic representation of FimH–uroplakin Ia (UP1a) interactions[97]. (a)
Originally, bacteria were interested in undergoing only weak interactions with the host cells
to retain mobility, a prerequisite to explore the urothelial surface for optimal nutrition
supply. For this purpose, the equilibrium between the low-affinity conformation,
characterized by an open binding pocket and intertwined domains (PDB ID: 4XOD), and
medium-affinity conformation, characterized by well-defined binding pocket (PDB ID: 4XOE),
is optimally suited. As urine flow arises, shear force induces a switch to high-affinity
conformation with an approx. 2000-fold[98] improved affinity, allowing the bacteria to now
withstand the shear force at least to a certain extent. In this conformation, the two domains
are pulled apart (PDB ID: 4XOB). When shear force ceases, FimH restores the medium-affinity
conformation. (b) When shear forces are low, bacteria can explore nutrition supply, and their
pili are in the slip-bond state. Once shear forces increase, bacteria are protected from
clearance, and the pili are in the catch-bound state. FL: FimH lectin, LD: lectin domain, PD:
pilin domain. Reprinted with permission from Mayer, K., Eris, D., Schwardt, O. et al. (2017)
Journal of Medicinal Chemistry 60: 5646–5662. Copyright 2017 American Chemical Society.
domain[96]. The two domains of FimH enable pathogens to induce a shift from low
to high affinity (catch-bond, Figure5.6)[97, 99]: Under tensile mechanical forces
induced by urine flow, FimH forms stronger interactions with uroplakin located on
the urothelial surface, preventing the elimination of bacteria by urination. More
recently, Sauer etal. demonstrated that the domain-separated state of FimH resulted
in a 2000-fold higher binding affinity compared to the domain-associated state of
FimH[98]. The relatively weak affinity of FimH in the absence of shear force in
turn enables bacterial motility on the urotheial surface, allowing rapid invasion of
new tissue areas[100, 101]. Because the catch-bond effect is not observed with the
domain alone, full-length FimH serves as the best target for antiadhesive
LD
FimH
drug screening. The first crystal structure of the FimH lectin was determined in
1999 for FimC–FimH complexed with an oligomannoside ligand [102]. Since then,
numerous structures of FimH (consisting of FimH
and FimHPD) alone or in com-
LD
plex with diverse mannoside ligands have been reported, greatly facilitating the
5.3 The Bacterial Adhesin FimH
Figure5.7 “Open” (left) and “closed” (right) conformation of the tyrosine gate. Left: crystal
structure of n-butyl mannoside (PDB code: 1TR7)[104] bound to the FimH CRD, as a
representative of the “open” conformation of tyrosine gate and “in”-docking mode of the
ligand. Right: crystal structure of biphenyl mannoside bound to the FimH CRD (PDB code:
3MCY)[108], as a representative of the “closed” conformation of the tyrosine gate and
“out”-docking mode of the ligand.
discovery of high-affinity FimH antagonists, such as the natural oligomannosides
(Man3 and Man9)[98, 103] and glycomimetics of various structures[97, 104–107].
The main features of FimH CRD can be summarized as follows: (i) A deep and
negatively charged pocket, the mannose moiety establishes direct hydrogen or
water-mediated hydrogen bonds; (ii) the entrance of the binding site, referred to as
the “tyrosine gate,” formed by three hydrophobic amino acids (Tyr48, Ile52, and
Tyr137), enables hydrophobic interactions with aliphatic/aromatic aglycones[35]
and (iii) the tyrosine gate can adopt two different conformations depending on the
ligand structures (open and closed conformation, Figure5.7)[103–105, 108].
145
;
5.3.3 FimH Antagonists
The different stages in the development of FimH antagonists are summarized in
Figure5.8. Already in 1979, Sharon and coworkers reported on the invivo activity of
methyl α--mannoside in a UTI mouse model[106]. In the following years, various
structural modifications with the main goal of improving the affinity were explored.
In 2005, Bouckaert etal. reported a series of alkyl α--mannosides (1, with n = 0–7)
as potent FimH antagonists[104]. As they could show on the basis of the X-ray of
n-butyl α--mannoside co-crystallized with FimH
, the potency is a result of van
LD
der Waals contacts of the alkyl aglycones with the so-called tyrosine gate, formed by
Tyr48, Tyr137, and Ile52. The best representative of the alkyl mannoside series,
n-heptyl α--mannoside, was used later on as reference compound. In addition to
alkyl groups, aromatic aglycones were explored to reach the hydrophobic tyrosine
gate. As early as the 1980s, Sharon and coworkers reported aromatic aglycones (→ 2)
to be able to enhance the binding affinity by a factor of approx. 600 compared to
methyl α--mannoside[109]. Such findings were later rationalized with squaric acid
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