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5.2 DC-SIGN-Mediated Viral Adhesion and Entry into Myeloid Cells
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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).
5.3 The Bacterial Adhesin FimH
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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
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.
nm wide, several micrometers long, rod-like fibers.
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)
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
FimH
domain alone, full-length FimH serves as the best target for antiadhesive
LD
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
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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
1 (REF. 104, 106)
7 (REF. 115) 8 (REF. 110) 9 (REF. 114) 10 (REF. 117)
2 (REF. 109)
3 (REF. 116)
Structural exploration
4 (REF. 108)
Prodrug strategyNew frontiers
5 R = Me (Prodrug)
6 R = Na (active principle)
(REF. 111)
Core structure
“Drug-like” properties
(PK/PD optimization)
Figure5.8 Structures of potent FimH antagonists and representative optimization strategies.
e.g. Target-directed
combinatorial library
(REF. 117)
FimH
5.3 The Bacterial Adhesin FimH
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derivatives (→ 3) and on the basis of various crystal structures (PDB code: 4AV5[103],
3MCY[108], 4CST[110], and 4CSS[110]) with biphenyl mannosides (→ 4, 8), indicating the positive effect of π–π stacking with the amino acid side chains of Tyr48 and
Tyr137 of the “tyrosine gate.” Furthermore, a favorable effect of ortho-substitution on
the aromatic ring adjacent to the anomeric center of up to a factor 10was reported by
several groups (→ 2, 5, and 6)[109, 111].
Further studies on FimH antagonists with extended aromatic moieties lead to a
series of modified biphenyl (→ 4-6, 8, 9)[108, 110–114], indolinylphenyl (→ 7)[115],
squaric acid derivatives (→ 3)[116], and pyrrolylphenyl (→ 10)[117], all showing
the affinities in low nanomolar range and oral availability.
For successful clinical applications of FimH antagonists, oral bioavailability and
fast renal excretion for reaching the targets in the urinary tract are prerequisites. In
2010, Klein etal. reported both the invitro and invivo PK data of a series of biphenyl
α--mannosides [111]. Because intestinal absorption and renal elimination are
related to opposed properties, i.e. lipophilicity for intestinal absorption and hydrophilicity for renal elimination, a prodrug approach was applied to meet both conditions. For example, after high intestinal absorption, the ester (→ 5) is hydrolzed by
esterases in the enterocytes and in the liver (“first pass”) releases the acid (→ 6),
which undergoes fast renal elimination to reach the target in the bladder, where it
realizes the therapeutic effect. Later structural modifications on FimH antagonists,
such as the indolinylphenyl derivative (→ 7)[115] or bioisosteric replacement of the
carboxylate [110], improved oral bioavailability in vivo. Among these modified
structures, the indolinylphenyls showed a high therapeutic potential, resulting from
optimized PK properties, and a substantial reduction of the dosage, i.e. a successful
1
treatment of UTI with a low dosage of 1 mg kg
without any additional administration of antibiotics. However, a major drawback of these indolinylphenyl antagonists
is their low solubility, limiting their further invivo applications. Therefore, a balance between solubility and permeability is another challenge for reaching oral bioavailability. Structural modifications, such as disruption of the molecular planarity
and introduction of heteroatoms, provided promising solutions to fulfill this criterion [117]. Finally, FimH antagonist GSK3882347 (structure not disclosed) has
already entered Phase I clinical study (NCT04488770) in a collaboration between
Fimbrion Therapeutics and GlaxoSmithKline.
147
5.3.4 Conclusion and Perspectives
Diverse strategies, besides traditional chemical synthesis in situ target-directed
dynamic combinatorial chemistry (Figure 5.8, New Frontiers) [107], were implemented in the search for high-potency FimH antagonists. Besides low-molecularweight antagonists, carbohydrate-based clusters of mannosides [118–122] and
carbohydrate dendrimers [123–126] were synthesized and extensively studied for
FimH inhibition[127]. Although these multivalent mannosides have shown high
potency, their sizes, polarity, and possible induction of gelation effects or hemagglutination invivo make therapeutic application unlikely.
5 Antiadhesive Carbohydrates and Glycomimetics
148
To summarize, in vitro and invivo functionally active mannosides block UPEC
adhesion through high-affinity binding to the mannose-binding site of FimH, thus
preventing bacterial colonization on urinary tract surfaces. Over the last decade, our
knowledge of FimH binding has been immensely expanded, thus facilitating the
rational design of FimH antagonists with diverse structural complexity. Structural
optimization for FimH antagonists has focused on improving pharmacokinetic and
pharmacodynamic properties, aiming at invivo potency and oral bioavailability.
Ideally, after oral administration, the mannosides, which are characterized by acid
stability in the stomach, will be quickly absorbed in the intestine and, once systemically available, will not be metabolized but rapidly eliminated by the kidney to reach
their therapeutic target, the type 1 pili in the bladder and urinary tract. Encouragingly,
some mouse models demonstrated that oral administration of the mannosides (→ 5,
7, 8, and 9) prevented UPEC colonization in the bladder in both acute and chronic
UTIs[110, 111, 114, 115]. Additionally, biphenyl mannoside (→ 6) has shown inhibitory potency on biofilm formation in vitro [114]. Recent research revealed that
FimH adhesins act synergistically with PapG-II adhesins–another virulence factor
prevalently expressed among the strains of UPEC causing pyelonephritis [128].
Therefore, in order to combat arising antibiotic resistance, combining both FimH
and PapG antagonists could lead to a more effective treatment for UTIs[129, 130].
5.4 Pseudomonas aeruginosa Virulence Factors
(PA-IL and PA-IIL)
5.4.1 Introduction
P. aeruginosa is an opportunistic Gram-negative pathogen that is part of the normal
flora in healthy adults. However, it can become lethally pathogenic in immunecompromised patients [131]. P. aeruginosa is involved in both acute and chronic
infections, especially in cystic fibrosis patients[131, 132]. The therapeutic options
for these infections remain limited because this pathogen exhibits increasing resistance to many antibiotics[133]. P. aeruginosa utilizes lectins and adhesins, exposed
on pili or flagella, for anchoring to the host cells. The soluble lectins PA-IL (or LecA)
and PA-IIL (or LecB) are expressed by P. aeruginosa, specifically binding to galacto-
sides and fucosides, respectively[27, 134, 135]. Both lectins are virulence factors
under quorum-sensing control but are, by themselves, cytotoxic to primary epithelial cells[136]. Natively, PA-IL binds to α-galactosylated glycosphingolipids in lung
epithelial cell membranes, while PA-IIL interacts with fucosylated and mannosylated epitopes but preferentially with Lewis
pneumonia model, both galactose and fucose reduced infection spread[138]. It was
also reported that human milk oligosaccharides significantly prevent adhesion of
P. aeruginosa to human respiratory epithelial cells[139]. In addition, a case report
described that combination therapy of tobramycin with both -galactose and
-fucose successfully cured an 18-month-old infant with systemic and pulmonary
infections caused by tobramycin-resistant P. aeruginosa[140]. Given an increasing
a
oligosaccharides[137]. In a murine
(a) (b)
Figure5.9 Crystal structures of PA-IL and PA-IIL in complex with d-galactose and l-fucose,
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respectively. (a) Binding sites of galactophilic lectin PA-IL complexed with d-galactose (PDB
code: 1OKO). Source: Adapted from Cioci etal.[135]. (b) Binding sites of l-fucose-binding
lectin PA-IIL complexed with l-fucose (PDB code: 1GZT). Source: Adapted from Mitchell
etal.[27].
percentage of antibiotic resistance, antiadhesive strategy alone or in combination
with antibiotics are expected to provide more effective clinical treatment to patients
suffering P. aeruginosa infections[141, 142].
2+
Both PA-IL and PA-IIL are tetrameric lectins that require Ca
ion for carbohy-
drate binding. PA-IL preferentially binds to terminal α--galactose in the presence
2+
of a bridging Ca
whereas PA-IIL binds unusually strongly (K
involvement of two Ca
ion in the CRD with a KD of 87.5 μM (Figure5.9a) [135, 143],
: 625 nM)[144] to -fucose with the
2+
ions (Figure5.9b)[27].
D
1495.4 Pseudomonas aeruginosa Virulence Factors (PA-IL and PA-IIL)
5.4.2 Mono- and Oligovalent Glycomimetic PL-Ligands
In nature, PA-IL displays high binding affinity toward α-linked galactosides, whereas
studies with synthetic glycomimetics demonstrated its binding preference for β-aryl
galactosides (Figures 5.10, 5.1 and 5.2) [145, 146]. Aromatic aglycones establish
favorable contacts within the CRD, e.g. the 2-naphthyl moiety forms a CH–π
“T-shape” interaction with His50, contributing to improved binding affinity[145].
When the aromatic aglycone of ligand 2 was presented tetravalently, even
nanomolar affinity could be determined by ITC (→ 3, Figure5.10)[147]. Diversified
multivalent scaffolds, such as tetravalent glycopeptide, glycoclusters of various
sizes, fullerene, and gold nanoparticles, have been introduced and evaluated[34].
To date, a rationally designed divalent galactoside (→ 4, Figure5.10) with a K
28 nM toward PA-IL has proven to be the ligand with the highest binding affinity so
far[148]. Another 1,3-alternated galactosylated glycocluster (→ 5, Figure5.10) with
of 176 nM against LecA has shown almost complete protection against
a K
D
D
of
1 (REF. 145)
5 (REF. 149)
2 (REF. 146)
4 (REF. 148)
3 (REF. 147)
Figure5.10 Representative structures of mono- and oligovalent PA-IL antagonists (1-5).
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