Sites for dynamic protein-carbohydrate interactions of O- and C-linked mannosides on the E. coli FimH adhesin

Article abstract of DOI:10.3390/molecules22071101

Antagonists of the Escherichia coli type-1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against acute and recurrent bacterial infections. In this study α-D-mannopyranosides O- or C-linked with an

Full text of DOI:10.3390/molecules22071101

molecules
Article
Sites for Dynamic Protein-Carbohydrate
Interactions of O- and C-Linked Mannosides on
the E. coli FimH Adhesin
Mohamed Touaibia 1,2,† , Eva-Maria Krammer 3,†, Tze C. Shiao 1, Nao Yamakawa 3,
Qingan Wang 1, Anja Glinschert 4, Alex Papadopoulos 1, Leila Mousavifar 1 , Emmanuel Maes 3,
Stefan Oscarson 4, Gerard Vergoten 3, Marc F. Lensink 3 , René Roy 1,* and Julie Bouckaert 3,
*
1
Pharmaqam, Department of Chemistry, Université du Québec à Montréal, P. O. Box 8888, Succ. Centre-ville,
Montréal, QC H3C 3P8, Canada; mohamed.touaibia@umoncton.ca (M.T.);
chichi_shiao@hotmail.com (T.C.S.); wange6988@yahoo.fr (Q.W.); usc.alex24@gmail.com (A.P.);
leilyanmousavifar@gmail.com (L.M.)
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB E1A 3E9, Canada
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), UMR8576 du CNRS, Université de Lille,
F-59000 Lille, France; eva-maria.krammer@univ-lille1.fr (E.-M.K.); Nao.Yamakawa@univ-lille1.fr (N.Y.);
emmanuel.maes@univ-lille1.fr (E.M.); Gerard.Vergoten@univ-lille1.fr (G.V.);
marc.lensink@univ-lille1.fr (M.F.L.)
2
3
4
Center for Synthesis and Chemical Biology (CSCB), University College Dublin, Belfield, Dublin 4, Ireland;
Anja.Glinschert@gmx.de (A.G.); stefan.oscarson@ucd.ie (S.O.)
*
Correspondence: roy.rene@uqam.ca (R.R.); julie.bouckaert@univ-lille1.fr (J.B.):
Tel.: +1-514-987-3000 (ext. 2546) (R.R.); +33-362-53-17-29 (J.B.);
Fax: +1-514-987-4054 (R.R.); +33-(0)362-53-17-01 (J.B.)
†
These authors contributed equally to this work.
Received: 1 June 2017; Accepted: 28 June 2017; Published: 3 July 2017
Abstract: Antagonists of the Escherichia coli type-1 fimbrial adhesin FimH are recognized as attractive
alternatives for antibiotic therapies and prophylaxes against acute and recurrent bacterial infections.
In this study α-D-mannopyranosides O- or C-linked with an alkyl, alkene, alkyne, thioalkyl, amide,
or sulfonamide were investigated to fit a hydrophobic substituent with up to two aryl groups
within the tyrosine gate emerging from the mannose-binding pocket of FimH. The results were
summarized into a set of structure-activity relationships to be used in FimH-targeted inhibitor
design: alkene linkers gave an improved affinity and inhibitory potential, because of their relative
flexibility combined with a favourable interaction with isoleucine-52 located in the middle of the
tyrosine gate. Of particular interest is a C-linked mannoside, alkene-linked to an ortho-substituted
biphenyl that has an affinity similar to its O-mannosidic analog but superior to its para-substituted
analog. Docking of its high-resolution NMR solution structure to the FimH adhesin indicated that its
ultimate, ortho-placed phenyl ring is able to interact with isoleucine-13, located in the clamp loop that
undergoes conformational changes under shear force exerted on the bacteria. Molecular dynamics
simulations confirmed that a subpopulation of the C-mannoside conformers is able to interact in this
secondary binding site of FimH.
Keywords: C-glycosidic linkage; ortho-biphenyl mannose; FimH; anti-adhesive; uropathogenic E. coli;
clamp loop; dynamic binding
1. Introduction
Pathogenic E. coli adheres via the FimH adhesin at the tip of their type-1 fimbriae to mannosylated
glycan receptors on epithelial linings [1,2]. The use of mannose-based anti-adhesives for the selective
Molecules 2017, 22, 1101; doi:10.3390/molecules22071101
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inhibition of type 1-pilus mediated bacterial adhesion [
3
–
24] has attracted great interest for the
non-antibiotic treatment of urinary tract [1,25] and intestinal [2,17,26] infections caused by pathogenic
Escherichia coli. These bacteria can express an arsenal of multiple adhesins, lectins with a variable
immunoglobulin fold, for their attachment to and colonization of host cells.
The α-D-mannopyranoside-binding lectin FimH recognizes, among other glycosylated receptors,
the multiantennary glycoprotein uroplakin Ia and attaches the bacteria to these receptors leading
to cystitis (bladder infections). The receptor-binding site of the FimH consists of a highly specific
mannose-binding pocket, with a tyrosine gate (Tyr48, Ile52 and Tyr137) at one extension and a
hydrophobic ridge (Ile13, Phe1, Phe142) at the other edge lining its entry [24,25] (Figures 1 and 2).
Figure 1. Crystal structures of O-alkylated
heptyl (green) in the closed tyrosine gate (PDB entry 4buq [13]), thioalkyl 37 (prussian blue, sulfur
atom in yellow) in the open gate (PDB entry 4avh [ ]) and butyl in the closed gate (apple green, PDB
entry 1uwf [ ]) and in the open gate (magenta, PDB entrz 1tr7 [ ]). ( ) The complexes have been
superimposed onto the FimH lectin domain complex with oligomannose-3 (almond white), bound in
the open tyrosine gate (PDB entry 2vco [ ]) and displayed on a hydrophobicity-colored surface (dodger
blue for the most hydrophilic, to white, to orange red for the most hydrophobic). Atom numbering
in the aglycons starts from the first carbon atom following the glycosidic O-linkage; ( ) The same
compounds are bound as in ( ), with potential van der Waals contacts and hydrogen bonds shown in
α-D-mannopyranosides as FimH antagonists, with aglycones
5
9
4
4
4
A
5
B
A
orange, dashed lines labelled with the inter-atomic distances in Å. W1 and W2 denote structural water
molecules important for the stabilization of the mannose that plugs into the mannose-binding pocket.
Figure 2. Compounds that only differ in its alkyne-phenyl order as bound in the FimH crystal
structures: 56 (blue, PDB entry 4av0) and its analog 61 (green, PDB entry 4auy). A wire wrapped
around the bond between carbon atoms at position two and three, or five and six, respectively in
compound 56 or 61, indicates the location of the alkyne. (A) Illustration of the overlay of the two ligand
aglycons with the C1-C2-C3 carbon atoms of the core mannose of N-linked glycans, displayed on a
hydrophobicity-colored surface presentation of the FimH lectin (dodger blue for the most hydrophilic,
to white, to orange red for the most hydrophobic). The atom numbering in the aglycons starts from the
first carbon following the O-glycosidic linkage; (
same color as the structure) are indicating contacts made by the 56-ligand (blue) in the open tyrosine
gate and by the 61-ligand (green) with the half-open conformations of Tyr48.
B
) Van der Waals contacts ( 4.5 Å, dashed lines in the
≤

Molecules 2017, 22, 1101
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The receptor-binding pockets in glycan-binding proteins often include tyrosine (and tryptophane)
residues with their aromatic side chains stacking against the apolar face of the pyranoside ring [27].
Such strong CH-
The open tyrosine gate conformation (Tyr48 side chain torsion angles near
the natural glycan receptor with highest affinity, oligomannoside-3, to pass in between the tyrosine
gate residues. The half-open conformation, reflecting a major change in the 1 torsion angle of the Tyr48
97 ), prevents the ligands from becoming sandwiched between the
]. Subsequent rotation around the 2 torsion angle of the Tyr48 side chain (near
π
stacking interactions also clamp oligomannoside-3 in the tyrosine gate of FimH [
5].
χ1 =
−
57◦, 2 = 83◦) allows
χ
χ
◦
◦
side chain (near
χ
1 =
−
164 ,
χ2 =
−
tyrosine si◦de chains [◦
9
χ
χ1 =
−
160 , χ2 = 10.8 ) closes the tyrosine gate. Thus far, the half-open and closed gate conformers have
only been observed in complexes with synthetic anti-adhesive molecules.
Due to its unique design, the FimH binding site accommodates rather well
α
-D-mannopyranosides
substituted with apolar aglycons, making these compounds very potent FimH inhibitors.
Mannopyranosides -substituted with heptyl [ 13 14 24 28] or with thiazolylamine [12 21 22 29
α
4
,
,
,
,
, , , ]
have been described as the most potent O-linked FimH ligands. However, O-linked compounds are
possibly susceptible to degradation by glycosidases. Low bioavailability and half-life of O-mannosides
can be attributed to the metabolic instability of the O-glycosidic bond [11
,18]) and the hydrolysis
by mannosidases [30 31]. Substitution of the O-glycosidic linkage by a C-glycosidic linkage was
,
recently shown to lend important advantages in the activity of mannose-based anti-adhesives
on the elimination of E. coli of intestinal cells [29] as well as for the treatment of urinary tract
infections [18
development [
substituents on
,
21]. para-Biphenyl derivatives O- and C-linked to
α
-D-mannopyranosides are under drug
32]. Based on the accumulated evidence that FimH interacts with hydrophobic
-D-mannopyranosides using its tyrosine gate [ 12 15 19 28], we set out to
6,
7,18
,
α
4,5,7,9, – , ,
determine specificity- and selectivity-enhancing structural parameters for the optimization of the
binding of drug-like anti-adhesive mannosides carrying either O- or C-glycosidic linkages to FimH.
In our search for challenging targets that might surpass the potency of known O-linked
mannopyranosides, we prepared
α-D-mannopyranoside derivatives with hydrophobic O- and
C-linked substituents (for an exhaustive list and the synthetic details, see Supplementary Information)
and sought for structure-activity relationships (SARs) explaining their binding to FimH. Amide,
sulfonamide, thioalkyl or aliphatic alkyl, alkenyl or alkynyl functionalities have been introduced
as spacers between the O- or C-anomeric atom of the glycosidic bond and the aglycons, in order to
enable interactions in the extended high-mannose glycan-binding pocket of FimH [5]. The solution
affinities of 55 mannosidic compounds with the E. coli FimH adhesin were measured using surface
plasmon resonance (SPR) detection and, where available, compared with solution affinities obtained in
isothermal titration calorimetry (ITC) measurements [9]. The mannosidic ligands with the most
promising affinities were further tested for their potential to inhibit bacterial adhesion using a
cellular assay, by inhibition of haemagglutination (HAI). The structural basis of the affinities was
examined by screening for interactions made between ligands and the FimH binding site using
available crystal structures and molecular simulation data of the complexes. As part of the SARs,
we present the high-resolution NMR solution structure of a high-affinity C-linked ortho-biphenyl
mannose derivative and its proposed binding modes to FimH, using induced-fit docking and molecular
dynamics simulations. The results of this study were used to construct a general view of the dynamic
binding modes of mannose-based anti-adhesives to the E. coli FimH. We hope this SARs study provides
a valuable vocabulary for the rational drug design of anti-adhesive molecules against uropathogenic
E. coli infections.
2. Results
2.1. Alkane Aglycons, the Nature and Dynamics of Their Interactions
A first series of compounds comprised alkylated
α-D-mannopyranosides as FimH binders.
Solution affinities of different mannosides for the FimH lectin domain (residues 1–158) have been

Molecules 2017, 22, 1101
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measured using SPR by competition of the soluble compounds with an immobilized receptor.
In initial measurements, the immobilized receptor was a Fab fragment derived from a monoclonal
antibody (IC10 (MedImmune, Gaithersburg, MD, USA), Kd = 220 nM) used previously to identify
the highest-affinity natural epitope for FimH (Kd = 20 nM for oligomannoside-3 (Figure S19) [33].
The measurements of the highest-affinity compounds were further refined by repeating the SPR
experiments using 8-aminooctyl α-D-mannopyranoside (5b) coupled onto the carboxymethylated
dextran of the sensor chip (CM5, Biacore3000, GE Healthcare Life Sciences, Marlborough, MA, USA)
via usual amide formation [34]. The steady-state affinity of 5b (Ss in Table 1) is determined by direct
binding of FimH to the mannose derivative coupled to the sensor chip and is almost identical to its
solution affinity of octyl α-D-mannopyranoside (5a, Kd = 21 nM, Figure S19), indicating that 5b can
be bound by the FimH lectin without steric hindrance from the sensor chip surface, thus providing a
simpler and effective bioanalysis.
Table 1. Summary of SARs on alkane aglycons: The solution affinity of the FimH lectin domain for
and O- and C-linked alkanes and thioalkanes. Ss: steady state affinity for 5b.
OH
O
OH
O
OH
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
R
O
S
O
35-39
106-110
4,5,5a,5b
S
R
R
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
R
Cpd
4
R
Cpd
Cpd
106
107
108
109
110
151 [4]
156 ± 45 [9]
100
16 ± 2.4
25 ± 6.3
H
35
36
37
38
39
–
–
–
–
21± 0.2 [4]
22 ± 2.7
22 ± 2.8
5a
5
–
–
–
–
–
–
4.3 ± 0.3 [4]
7.3 ± 1.8 [9]
6.25
14 ± 5.0
36 ± 9.0
59.5 ± 4.7 [9]
–
–
25
18 ± 0.4
Ss 17 ± 0.4
–
14 ± 7.6
78 ± 5.6
5b
–
–
–
–
NH2
11 ± 6.1
32 ± 4.1
–
–
–
–
Compounds 35
weaker binders than O-mannopyranosides with plain alkyl chains (compounds
(hepta) and 38 (hexa) vs. heptyl (compound ) and hexyl (Kd = 10 nM) [ -D-mannopyranosides
(Table 1). Interestingly, ITC and HAI measurements confirmed that heptyl -D-mannopyranoside
–39 including a sulfur atom in the alkyl chain of O-mannosides were found to be
4–5), for example 37
5
4] α
α
5
exhibited inhibition potency that was ~4-fold higher than its 4-thio analog 37. This observation
provided validation for the experimental methods in this affinity range. A property shared by
alkane-substituted O-(5, 37) and C-linked (107) mannopyranosides is the appearance of an optimum
affinity for those mannosides with a chain length of seven atoms beyond the glycosidic linkage (Table 1).
This length coincides with the optimal binding interactions of the natural oligomannose-3 receptor in
the tyrosine gate of FimH (Figure 1).
The overlay of mannosides bound in the crystal structures shows how far the compound structures
reach beyond the
α1,3-linked mannose bound in the mannose-binding pocket of FimH and how they
can mimic contacts of the oligomannoside-3 N-glycan receptor with the FimH protein (Figure 1).

Molecules 2017, 22, 1101
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The alkyl substituents follow the hydrophobic “B”-side (C1-C2-C3) of the core mannose (Figure 1A).
Together with the C2-C3 bond of the 1,3-linked mannose, carbon atoms two and three of the alkyl
interact with the Ile52 side chain (Figure 1B). At atomic position four, the alkyl chains diverge for
making CH- interactions with either one of the tyrosine side chains. In the open gate, the atom at
position four prefers Tyr137, as seen for the end-standing methyl group of (magenta) and the sulfur
atom of 37 (prussian blue). In the closed tyrosine gate, the interactions are predominant with the
extended conformation of the Tyr48 side chain for the butyl (apple green) and the heptyl (green)
-D-mannopyranosides. In summary, ligands in the open gate interact more intensely with Ile52 and
α
π
4
4
5
α
Tyr137, whereas residues in the closed gate interact more strongly with Ile52 and Tyr48. The first two or
three atoms beyond the glycosidic oxygen interact with the Ile52 side chain.
In the crystal structure of FimH in complex with 37 [9], the electron-rich sulfur atom is polarized
in its interaction towards the Tyr137 side chain. It contributes to interactions in the hydrophobic gate
similar to the Manβ1,4GlcNAc glycosidic linkage in oligomannoside-3, namely four atoms beyond the
glycosidic linkage (yellow, dashed line in Figure 1B). Carbon atoms at position five, six and seven after
the glycosidic linkage of 37 (prussian blue) follow the C5-C6-O6 bond of the trimannose core-linked
GlcNAc and stack with Tyr48 of the open tyrosine gate (Figure 1A). The O-linked alkyl mannosides
presented thus mimic relatively well the natural oligomannose-3 receptor binding with the FimH
lectin, also when they contain the sulfur at anomeric position four beyond the glycosidic linkage.
For C-linked mannopyranosides 106–110 (Table 1) in contrast, the sulfur atom in the corresponding is
positioned one atom earlier and would fall shortly near Ile52 (in the open gate conformer) or Tyr48 (in
the closed gate) (Figure 1B). Its repulsion might have given rise to slightly lower affinities. This was
indicative that a heteroatom’s position in the aglycon and thus in the tyrosine gate determines its
translating into affinity. Bulkier and electron-rich atoms appear better positioned between positions
four or six beyond the glycosidic linkage, but not on position two or three where they might undergo
repulsion by either Ile52 or Tyr48. The following sections, detailing FimH affinities for compounds
bearing alkane, alkene and alkyne linkers between the mannose and aromatic substituents, provide
further evidence for this.
2.2. An Alkene, But Not an Alkyne, Linker Increases Affinity by Interaction with Ile52
The O-linked mannopyranosides 22
FimH inhibitors than their hydrogenated alkane analogs 26
for 23 25 make these compounds at least equipotent to the reference FimH antagonist heptyl
-D-mannopyranoside . It appears that the apolar interactions created between the double bond
of the O-linked mannosides 22 25 and FimH contribute more favorably to the affinity than those
–
25 carrying an alkenyl aglycon were found to be better
–
29 (Table 2). Kd values below 5 nM
–
α
5
–
created by a single bond. The double bond is made between atom positions two and three beyond
the glycosidic linkage. These carbon atoms are most likely to make hydrophobic contacts with Ile52,
a residue important in oligomannose-3 binding (Figure 1). Next we examined whether the affinity
could be further improved by the introduction of a more rigid alkynyl linker between the aromatic
moiety and the anomeric oxygen of mannose (Tables 2 and 3).
Among alkyne, alkene and alkane-substituted O-mannopyranosides, the alkene with its double
bond appears most suitable for inhibition of FimH (Table 2). Nevertheless, the HAI data indicated that
alkanes are equal (compound 27) or even 2-fold better (compound 29) in inhibitory potency than the
alkenes (compounds 23 and 25, respectively) (Table 2 and Table S2). Alkyls are slightly less soluble
than alkenyls and it is presumable that the inhibition potential by 27 and 29 has been slightly hampered
by the lack of DMSO in the SPR experiment.

Molecules 2017, 22, 1101
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Table 2. Affinities of FimH for O-linked alkene-(22–25), alkane-(26–29) and alkyne-(50–53) derivatized
O-mannopyranosides, as measured in SPR and/or ITC experiments, and their minimal concentrations
inhibiting haemagglutination (HAI).
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
Kd SPR (nM)
Kd ITC nM)
HAI (µM)
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
R
Cpd
22
Cpd
26
Cpd
50
53 ± 7.5
59 ± 8.5
168 ± 15
–
–
–
–
–
–
3.0 ± 1.1
10 ± 0.2
405 ± 24
23
24
25
–
27
28
29
1.5 ± 0.3
51
52
53
61 ± 9.6
25
25
100
4.3 ± 0.8
13 ± 1.7
2250 ± 291
–
–
–
–
–
–
5.0 ± 1.4
22 ± 3.0
120 ± 18
71 ± 23
–
–
50
25
50
Table 3. The affinity of the FimH lectin domain for alkyne O-mannopyranoside series 41
56: PDB entry 4av0) and two compounds with an inverted aglycon structure compared to 56 61 (PDB
entry 4auj) and 62. * nf: SPR data could not be fitted, see also Figure S20.
, 54–57
(
:
OH
O
HO
HO
HO
OH
O
HO
HO
HO
O
R
61, 62
O
41, 54-57
OR
Cpd
41
R
Kd SPR (nM)
Kd ITC (nM)
HAI (µM)
H
490 ± 55
–
–
54
55
133 ± 31
–
–
S
83 ± 5.6
105 ± 34
50
56
57
53 ± 2.3
104.6 ± 21.9
50
–
816 ± 105
–
61
62
H
36.9 ± 3.0
18.3 ± 5.9
59.5 ± 2.3
12.5
25
Me
nf *

Molecules 2017, 22, 1101
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Although the enhanced affinity of the alkenes over the alkanes may be negligible, it is directed
by specific recognition events at post-glycosidic linkage atomic positions two to three with Ile52 and
Tyr48 of the tyrosine gate (Figures 1B and 2B), rather than singly by a hydrophobic effect. The alkynyl
inhibitors 50
alkyl-linked compounds 26
–
53 were much less active than the alkenyl linked mannopyranosides 22
–25 and the
–
29. In contrast to the alkenyl mannopyranosides, the rigidity of the sp1
hybridization of the alkynyl linker is likely to impede significantly the positioning of the aglycon in its
most preferred binding conformation. This rigidity effect dominates over possible positive dispersion
interactions between the alkynyl linker and the hydrophobic gate.
The substitution of the phenyl group of the alkyne derivative 50 (Kd = 168 nM) by either para
(51) or ortho (52) biphenyl showed improved potency (Table 2). The introduction of naphthalene (53,
Kd = 120 nM), 2-thiophene (54, Kd = 113 nM), 3-pyridine (55, Kd = 83 nM) and a para-methoxyphenyl
group (56, Kd = 53 nM) also progressively improved the affinity (Table 3). A nitro group at the para
position of its phenyl group (57, Kd = 816 nM) significantly diminished the affinity compared with 56
,
most probably because this electron-withdrawing group decreases its stacking potential with Tyr137
(described in [13]). Compounds 56, 61 and 62 each contain an aromatic ring and a propargyl group in
their apolar tail, but with inverted order of spatial disposition for these functional groups. Among
these three compounds, the affinity is highest for 61. In the co-crystal structure of 61 bound to the FimH
lectin domain (PDB entry 4auj), the ligand sterically clashes with Tyr48 in its open gate conformation:
the bulky phenyl group of 61, juxtaposed to its glycosidic linkage, dislocates the Tyr48 side chain
and forces it into dynamic alternative conformations such as the half-open tyrosine gate conformation
(Figure 2) [9,13].
The trend of increasing affinity of FimH for compounds 61
measurements is confirmed in the HAI assay and by affinities previously obtained in ITC
measurements [ ] (Table 3). In order to correlate the affinity of 56 and 61 with their binding mode
to FimH, these compounds had been soaked into crystals of ligand-free FimH in the trigonal space
group (PDB entry 4auu) [ ]. These crystals have the important advantage that no crystal packing
> 62 > 56 obtained in SPR
9
9
contacts are present at the level of the tyrosine gate. Therefore, a compound that binds into the tyrosine
gate of such FimH crystals experiences similar degrees of freedom as in solution. The thus obtained
crystal structures of FimH show that the two aglycons indeed stack very differently in the tyrosine gate
(Figure 2). Compound 61 also shows more potential for making close hydrophobic contacts (Figure 2A)
when compared with 56 (Figure 2B), supported by its higher affinity for FimH (Table 3). The terminal
hydroxyl group of 61 is in contact with the solvent, where it is coordinated in a hydrogen-bonding
network via a water molecule to the hydroxyl groups of Tyr48 and Thr51. The observed hydrogen
network cannot be formed by compound 62 that bears a methoxy instead of a hydroxyl group. This
might be the structural reason behind the 2–3 fold improved affinity of compound 61 over 62 (Table 3).
2.3. Sulfonamide and Amide Spacers: Worse Fit and Bulky Compensation
Next, mannopyranosides with sulfonamide- and amide-based spacers were tested and the nature
of the glycosidic aglycones was changed from O- to C-linkages. Several interesting SAR trends could
be derived from this series (Table 4). The O-linked sulfonamides (70–75), just like the alkynyl in ligands
41 and 50 57 (Table 3), introduced stiffening just beyond the O-glycosidic linkage. This prohibits
–
an arbitrary positioning of the ligand in the tyrosine gate according to best complementarity and
may explain why the ligands profit from increased interaction possibilities through an increase in
size and polarizability (Table 4). For example, there was a significantly improved potency for the
tri-isopropyl-substituted phenyl derivative 74 (Kd = 37 nM) that has its phenyl ring extended with
bulky hydrophobic groups. Replacing the phenyl with a naphthalene group did not increase the
affinity (75, Kd = 148 nM), and the insertion of the electron-withdrawing nitro group had the opposite
effect (71, Kd = 408 nM) (Table 4).

Molecules 2017, 22, 1101
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Table 4. The affinities of FimH for O-linked mannopyranoside sulfamides 70–75 and their C-linked
mannopyranoside amide analoges 91, 92, 93 and 94, 95 and 96, as measured using SPR detection.
OH
O
HO
HO
HO
HO
OH
O
HO
HO
O
O
O
S
70-75
91-96
O
N
R
H
NHR
Cpd
70
R
Kd (nM)
Cpd
73
R
Kd (nM)
Cpd
91
R
Kd (nM)
Cpd
94
R
Kd (nM)
146 ± 26
122 ± 11
372 ± 26
227 ± 54
71
72
408 ± 52
168 ± 64
74
75
37 ± 5
92
93
397 ± 83
105 ± 44
95
96
328 ± 63
148 ± 18
970 ± 382
We discovered that the FimH affinity towards C-linked mannosides 91–96 could be improved by
the insertion of a flexible alkyl spacer between the phenyl ring and the amide function and that the
length of this alkyl spacer was important. Indeed, while mannoside 92 (Kd = 397 nM) that contains
a methylene spacer was still as potent as the aromatic amide 91 (Kd = 372 nM), the FimH inhibitory
capacity rose 4-fold for 93 (Kd = 105 nM) carrying a propyl spacer thus placing the aromatic moiety at
the sixth position from the C-glycosidic linkage. The introduction of an ethyl linker had no enhancer
effect (96, Kd = 970 nM).
2.4. C-Mannopyranosides versus O-Mannopyranosides
In accordance with recent studies reporting that the FimH binding affinity and pharmacological
characteristics of alkyl- and aryl -D-mannopyranosides could be further improved by the insertion of
α
extra substituents [ 16 17 19 26,29] substituting the para and ortho positions of the phenyl ring with an
9,
,
,
–
additional phenyl ring or inserting a naphthalene group most frequently surpassed the binding affinity
of the unsubstituted phenyl derivative (Table 5). The inhibition potential of 116 and 117 exceeds the
one of their parent 115 some 4- and 26-fold, respectively, indicating additional interactions of the extra
phenyl substituent in the tyrosine gate.
The affinities for the C-linked compounds bearing amide linkers (Table 4) can be directly compared
with analogs with alkene or alkane linkers (Table 5), because of their identical R-group in the aglycons.
For example, the alkene-linked compound with a phenyl group, 115 (Kd = 182 nM), is 2-fold more
active than its amide-linked analog 91 (Kd = 372 nM). We examined whether an alkane instead of an
alkenyl spacer would increase the potency further. Three of the five aryl C-mannopyranosides (119
120 122) with alkyl linkers have better affinities than their corresponding alkenyl parents and HAI
data confirmed this trend (see also Table S2).
,
,
For these compounds, the more limited conformational freedom of the double bond in the alkenyl
mannopyranosides, relative to the single bond in the alkyl mannosides, may actually hinder the aryl
moiety from occupying its preferred position in the hydrophobic groove of the FimH binding pocket.
The crystal structures of FimH revealed that carbon atoms at positions two and three beyond the
glycosidic atom make van der Waals contacts with Ile52, explaining why the double-bond character of
this position would be beneficial (Figure 1).
The alkenyl occurs one atomic position earlier in our C-linked compounds, thus possibly
generating less van der Waals contacts with Ile52, or pulling bulky aromatic groups deeper inside
the tyrosine gate. Therefore, the affinities of alkenylated C-linked mannopyranosides for FimH are

Molecules 2017, 22, 1101
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generally a bit lower than for their O-linked analogs, for example the affinity of the alkenyl phenyl
(115, Table 5 vs. alkyl phenyl (22, Table 2) is indicative of the latter.
Table 5. The affinity (using SPR) of FimH for C-linked mannopyranosides 78
, 115–118, 119–123. nf: no
good fit could be obtained of the data to the Langmuir 1:1 binding model (Figure S20).
OH
O
OH
O
HO
HO
HO
HO
HO
HO
78, 115-118
119-123
R
R
R
Cpd
115
Kd (nM)
Cpd
119
Kd (nM)
182 ± 25
49 ± 5.3
116
117
17 ± 3.5
6.9 ± 5.7
120
121
nf
123 ± 21
118
78
nf
122
123
nf
H
118 ± 20
266 ± 26
The 117/121 alkenyl/alkyl pair is an exception to this trend. This may be due to the different
and specific interactions of 117 with FimH. The ortho-substituted 117 (Table 5) demonstrated an
in vitro affinity similar to its O-analog 24 (Table 2). We decided to further explore the structural basis
for its successful design using NMR spectroscopy, crystal structures, rigid and induced-fit docking
approaches and molecular dynamics (MD) simulations.
2.5. Ortho-Biphenyl C-Mannopyranoside 117
The three-dimensional structure of 117 was determined using high-resolution (900 MHz) NMR
in DMSO-d6 solution. This C-linked analog is in the usual 4C1 conformation as judged by the total
lack of nOe between H1 and H4/H6, together with strong nOe between the germinal aglycon Ha,b
protons with the axial H3 and H5 protons (Figure 3A,B, Table S1, Figures S14–16). The NMR-derived
structure of C-linked ortho-biphenyl ligand 117 was superposed onto the O-mannopyranoside of 61
(PDB entry 4auy) bound into the mannose-binding pocket of FimH that features both the open and
half-open tyrosine gate conformers (Figure 3C) [9]. This revealed an as yet unexplored secondary
mode of binding onto FimH, where its first phenyl ring interacts with Tyr48 and its second phenyl
ring in the ortho-position docks near the isoleucine 13. Ile13 is located at the lower hydrophobic
ridge of the mannoside-binding pocket of FimH, also called the clamp loop because it undergoes
major conformational changes when FimH forms high-affinity catch bonds with mannosides [28].
Interestingly, this binding position was not retrieved as a low energy binding pose in different docking
trials in which the binding site residues were either fully flexible or in which the tyrosine gate was held
open, half-open or closed. Fully flexible docking always led to low-energy binding poses in a half-open or
almost closed tyrosine gate, implying that Tyr48 makes an aromatic stacking interaction with the first

Molecules 2017, 22, 1101
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phenyl ring of 117 (Figure 3E). The observed discrepancy between the docking and the NMR results
led us to perform MD simulations.
Figure 3.
mannopyranoside. Quaternary carbons were attributed with the help of HMBC experiments; (
Conformation of 117 at 293 K in DMSO-d6; ( ) The NMR-derived solution conformation of 117
superposed in FimH of PDB entry 4auy; ( ) Superposition of the simulated minor conformer of 117
(green), as extracted from the MD simulation without internal structural waters (minor, 11%), with
PDB entry 5abz (salmon) presenting two alternative binding modes of a naphthyl mannoside; ( ) Best
solution for the binding of 117 from induced fit docking and ( ) comparison with the major binding
(A) Proton assignments based on NOESY and TOCSY, for compound 117 C-linked
B
)
C
D
E
F
modes as simulated using MD, for 56% and 61% of the time in the form with the internal structural
waters included (green) or not included (blue), respectively.

Molecules 2017, 22, 1101
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The best-scoring pose from the induced fit docking (Figure 3E), as well as the best-scoring poses
from rigid dockings performed on open, half-open and closed tyrosine gate conformers, were subjected
to MD simulations. Independent of the initial binding pose, the mannopyranoside of 117 always
remained positioned in the same position and orientation in the MD trajectories. The non-mannosidic
aglycon part was dynamic and featured four binding modes (Figure S21). Interestingly, the closed/Tyr48
conformer was very similar to what is observed for the best-scoring docking solution using induced-fit
docking (Figure 3E) and the NMR-derived solution structure of 117 turned out to simulate well the
closed/Ile13 binding conformer (compare Figure 3C with Figure 3D). However, visual inspection
clearly pointed out that the closed/Ile13 ligand conformer was minor in presence and being visited
only glancingly throughout the simulations. A possible explanation could be that the NMR solution
structure of 117 was determined in DMSO and not in water. A simulation performed without the
structural waters clearly highlighted that as long as these structural water were absent, the ligand was
close to Ile13 (Figures 3D and 4D) and that only after the water (W1, Figure 1B) was able to penetrate
into the mannose-binding pocket, the ligand aglycon part orients itself towards the tyrosine gate
(Figure 3F, movie provided in Supporting Information).
3. Discussion
Mammals possess many different types of α-D-mannopyranoside-binding lectins and thus target
selectivity needs to be cautiously addressed [35]. Exposed at the edge of the type-1 pilus on the
bacterial envelope, the FimH adhesin is maintained in a low-affinity conformation by interactions of
the lectin domain with the pilin domain of FimH [36]. These quaternary interactions are disrupted
when the bacteria adhered to a mannosylated surface and experience shear stress and consequently the
FimH mannose-binding lectin domain is extended and forms catch bonds with the mannosides [37].
Despite that the crystal structures of the isolated lectin domain always contain the high-affinity
FimH conformation, the binding site of FimH remains dynamic to enable the accommodate of a
plurality of mannosylated receptors [24]. Tyr48 and Tyr137, together with Ile52, form the so-called
tyrosine gate [
multiple conformations depending on ligand structure [
4
]. Their side chains are entropic adaptors to ligand structural changes and can adopt
]. Indeed, several recent crystal structures
9
of FimH complexes show the binding of hydrophobic aglycons substituents that fit poorly in the
tyrosine gate and remain highly dynamic. Sometimes a high affinity can be acquired through such
a dynamic binding, for example for compound 61, where the interaction energy is mainly driven by
dispersion [13]. In one of the two FimH molecules of the asymmetric unit of a crystal structure of the
lectin domain in complex with a C-linked naphtyl mannose analog (PDB entry 5abz) [38], at least two
alternative aglycon conformers were observed: a first one was bound between the two tyrosines in the
open gate, whereas a second one hovered over the hydrophobic ridge above Phe142 (Figures 3D and
4D). The same binding mode was found in docking experiments using bivalent mannosidic inhibitors,
posing one arm in the open tyrosine gate and one arm above the hydrophobic ridge [39].
Derivatives of α-D-mannopyranoside with O- and C-glycosidic linkages bind the FimH adhesin,
a mannose-specific lectin located at the tip of type 1 pili of pathogenic E. coli, and thereby inhibit
bacterial adhesion. The structural basis of the affinities with the interactions made between ligands
and the FimH binding site was examined using crystal structures of the complexes, which allows
the construction of an overview of the dynamic binding modes of mannose-based anti-adhesives
to the E. coli FimH adhesin. Alkyl and aryl derivatives of
α-D-mannopyranoside with O- and
C-glycosidic linkages can establish stacking interactions with the hydrophobic residues surrounding
the mannose-binding pocket of the FimH adhesin of type 1-piliated uropathogenic E. coli. In earlier
work, a para-biphenyl
compound 61, was found to bypass the tyrosine gate and establish an aromatic stacking with Tyr48
on the outside of the tyrosine gate (PDB entry 3mcy, Figure 4C) [ ]. With the structural studies of
the ortho-biphenyl substituted C-linked mannopyranoside 117 (Figure 4D), more evidence is brought
α-D-mannopyranoside with a short, flexible O-linkage, similar to the linker in
7

Molecules 2017, 22, 1101
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forward that the hydrophobic ridge exposing Ile13, Phe1 and Phe142 may also play an role as a
secondary binding site in dynamic interactions of the FimH lectin with oligomannosylated receptors.
Figure 4. Different modes of binding to FimH. The way the carbohydrate-based inhibitors interact
with the FimH lectin domain can be classified in major (
Tyr48-loving: PDB entries 4avh (beige), 4auy (blue) and 5mts (fuchsia); (
4av0 (blue), 1tr7 (beige); ( ) bypassing the tyrosine gate: the ligand para-biphenyl
entry 3mcy, fuchsia) and a high-scoring docking pose of the ligand in PDB entry 5mts (beige), that
both stack on the other side of Tyr48, outside the tyrosine gate; ( ) sampling of aromatic substituents
A
,B
) and two minor (
) Tyr137-loving: 4av5 (fuchsia),
-D-mannose (PBD
C,D) binding modes (A)
B
C
α
D
C-linked to mannose near Ile13 of the clamp loop, that traps the internal water molecule hydrogen
bonding to the axial O2 hydroxyl of mannose: PDB entry 5abz (beige) and a MD cluster (11% of the
total, when the internal structural water molecule was absent at the start of the simulations) of the
ortho-biphenyl α-D-mannose 117.
Only C-linked mannospyranosides derivatized with a para-substituted biphenyl have been further
explored so far, both for their SARs [22
Synthetic C-glycosides most often display a reduced activity compared to their O-linked parents [31].
However, C-glycosides are of higher stability, because they are not metabolized by the host enzymes [18 29 30].
Earlier comparisons of the bioactivities of C-glycosides with those of their O-glycosylated parents have
uncovered several candidates with diminished or similar activity [ 18 29 41], but none exhibiting
a significant improvement. Modification of the O-linked -anomer by a C-linkage leads to a loss
in polar character and thus hydrogen bonding capacity [29 38]. In the synthetic work presented
,38,40] and their pharmacological characteristics [21–24].
,
,
8,9, , ,
α
,

Molecules 2017, 22, 1101
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here, it also implies a pharmacophore position shifting. In our studies, the affinities of the C-linked
mannopyranosidic derivatives turned out to be relatively similar to their O-analogs. The ortho-biphenyl
substituted C-linked mannopyranoside 117 constitutes a potent C-linked mannopyranoside derivative.
Remarkably, the high-resolution NMR solution structure of 117 can be superposed in an active binding
mode to a secondary site (including Ile13) that agrees with a minor binding mode from our MD
simulations. The conformer adopted by the hydrophobic compound 117 when solubilised in DMSO-d6
can thus right away occupy an unusual hydrophobic patch on FimH guided by the Tyr48 and Ile13
residues in the hydrophobic surrounding of the FimH mannose-binding pocket. The ortho-biphenyl
substituted C-linked mannopyranoside 117 has a higher affinity over the para-substituted analogue
116. This can be explained by its particular binding modes: (1) the first phenyl ring stacks with the
Tyr48 side chain; (2) the ortho-placed second phenyl ring interacts either with Tyr137 or with Ile13.
Even so the simulations indicate that the position of 117 towards Ile13 is a minor conformation and is
likely to occur in an early step of the binding event, before the waters can occupy their final place and
being held in a hydrogen bonding network, this conformation is of particular interest as the change in
conformation of the Ile13-containing loop is associated with shear-force enhanced bacterial adhesion.
Binding of anti-adhesives at the clamp loop holding Ile13 where large shear-force induced
conformational changes occur, with the interplay of aglycon stacking with either the Ile13 or the Tyr48
side chain, is a mechanism as yet little explored in drug discovery of FimH antagonists of E. coli
adhesion [42]. These novel structural insights, namely that ortho-biphenyl substituents on mannose can
sample a binding interface on the FimH adhesin, notably one that is directly involved in the regulation
of FimH affinity of pathogenic E coli under shear force, opens new perspectives for non-antibiotic
therapies and anti-adhesive design [39,40,43].
4. Materials and Methods
4.1. Solution Affinity of Ligands for the FimH Adhesin Using SPR
4.1.1. Steady State Affinity Measurement
The FimH lectin domain (Phe1-Thr158 of the fimh gene product of E. coli strain J96) was expressed
and purified as described [5]. Affinity measurements using surface plasmon resonance (SPR) were
performed on a Biacore 3000 as described earlier [4,33]. The equilibrium dissociation constant Kd
of FimH for the mannose derivatives was determined using a competition assay in solution. In this
assay, the mannose derivatives compete with an immobilized Fab fragment from the monoclonal
anti-FimH antibody 1C10 or with an immobilized 8-aminooctyl α-D-mannopyranoside (5b) for FimH
binding, as described earlier [34]. The global kinetic constants ka, kd and Rmax of the FimH-ligand
interaction of binding were fitted using the BiaEval software. Subsequently, a constant concentration
of FimH that was close to the Kd of the FimH-ligand interaction was added to a concentration series
of mannoside (2000 nM down to 1.95 nM) and flown over the Fab-immobilized or 8-aminooctyl
α-D-mannopyranoside (5b) modified biosensor chip (Figure S18).
4.1.2. Competition Method
The competition binding assay measures the amount of free FimH in solution using biosensor
chips decorated with immobilised Fab fragments of a monoclonal antibody or with an immobilized
8-aminooctyl
α
-D-mannopyrannoside (5b). [FimHnb] = [FimH0]
−
[FimH-Man] (where [FimHnb] is
the concentration of non-bound fraction of FimH, [FimH0] the intitial, total FimH concentration and
[FimH-Man] the mannoside-bound FimH concentration). This amount of free FimH, measured by
the SPR assay, is proportional to the dissociation constant for the FimH-mannoside binding. These
concentrations of FimH unoccupied and able to bind to the ligand on the chip were then fitted
by feeding the 1:1 Langmuir binding model, keeping the parameters ka, kd and Rmax measured in
the foregoing kinetic binding (to IC10 or 5b) constant. For improved accuracy, two overlapping

Molecules 2017, 22, 1101
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2-fold dilution series of the mannoside derivatives were used to allow a smaller than 2-fold dilution
concentration series and abundous data points. Using two parallel series 2-fold dilutions to create
concentrations that were only 1.5-fold different, from 2000 nM down to 10 pM of mannoside derivatives,
for most of the compounds, inhibition curves could be fitted to the equation (Figure S19), with some
exceptions (Figure S20).
4.2. Haemagglutination Inhibition Assay
The SPR assay was not optimal for those mannosides with lower solubility because of incompatibility
with the use of DMSO for the measurement of nanomolar affinities. Therefore we have cross-validated
the compounds that displayed solubility issues and their analogues with results from another molecular
assay (ITC) [9,13] and have performed one cellular assay, by haemagglutination inhibition (HAI).
The inhibition of guinea pig red blood cell agglutination by the type-1 piliated uropathogenic E. coli
strain UTI89 was evaluated in a 2-fold dilution series of the mannosides. UTI89 E. coli were grown
statically overnight at 37 ◦C in LB (per liter of water: 10 g tryptone, 5 g yeast, 5 g NaCl). The bacterial
pellet was washed by centrifugation and diluted in buffer (Na-Hepes 20 mM, NaCl 150 mM, pH 7.4)
before determination of it haemagglutination titer. The mannosides (25
series in the same buffer as the bacteria, however 10% DMSO was maintained in the final volume
(100 L) of each well. The bacterial solution (25 L) was added at a concentration of four times the
bacterial haemagglutination titer. Finally, guinea pig red blood cells (50 L), washed in buffer and
µL) were diluted in a 2-fold
µ
µ
µ
diluted to 5%, were added and allowed to stand for 1 hour at 4 ◦C before read-out of the inhibition
titer: the highest concentration of mannoside that can still inhibit the haemagglutination by bacteria.
The titers for the mannosides are here expressed in µM.
4.3. Induced Fit Docking
Docking experiments were performed with the GOLD software (The Cambridge Crystallographic
Data Centre, Cambridge, UK). The six heavy atoms of the mannose ring found in the coordinate file
(PDB entry 4auy) were used as a scaffold in the active site. Two series of docking procedures were
performed, with a rigid and a fully flexible ligand-binding site. The following residues in the binding
site vicinity: Asp47, Tyr48, Ile52, Thr53, Asp54, Gln133, Thr134, Asn135, Asn136, and Tyr137 were also
defined as flexible in the latter. For each ligand, 10 poses that were energetically reasonable were kept
while searching for the correct binding mode of the ligand. The decision to keep a trial pose was based
on a computed energy for the interaction of the ligand with receptor of that pose. The ChemPLP fitness
scoring function is the default in GOLD version 5.2 used to rank poses. Additionally an empirical
potential energy of interaction
E(interaction) = E(complex)
Emperical Potential Energy Function SPASIBA and the corresponding parameters are used [46
∆
−
E for the ranked complexes was evaluated using the simple expression
(E(protein) + E(ligand) [44 45]. For that purpose, the Spectroscopic
47].
∆
,
,
Discovery Studio Visualizer 4.1 (Accelrys, San Diego, CA, USA) was used for viewing, Chimera version
1.8 (University of California, San Francisco, CA, USA) for figure preparation.
4.4. Molecular Dynamics Simulations
The best scoring complex of compound 117 and FimH from the induced fit docking with either
the tyrosine gate helt open (system 1a), closed (system 2) or half-open (system 3) were used as the starting
conformations for the MD simulations. The complex was solvated and the structural waters were
added. The ionic concentration was set to 0.15 M NaCl. In accordance with propKa [48], the standard
protonation state at pH 7 was used for all protonatable groups of FimH. The generated molecular
system comprised about 45,000 atoms and had a size of about 66
×
90
×
70 Å. The CHARMM36
force field with CMAP corrections was used to describe protein, water, and ion atoms [49]. Missing
force field parameters for compound 117 were initially generated with CgenFF [49] with standard
parameters and afterwards adapted. The integrity of the compound was verified in a 50-ns long MD
simulation of the compound alone in water using the adapted force field. Two independent simulations

Molecules 2017, 22, 1101
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of the generated system were performed. In each of them, a three-step equilibration was applied:
first, a 2.5-ns long equilibration of the water and ions molecules, second a 2.5 ns long equilibration
in which only the protein backbone was fixed, and third unrestrained simulations was carried out
for 2.5 ns. This was followed by a 30-ns long production run. In addition, a single simulation was
performed using the same protocol as described above with the system 1a only without placing the
structural waters (system 1b). All MD calculations were performed in the isothermal-isobaric ensemble
at 300 K with the program NAMD2.9 [50] using periodic boundary conditions. Long-range electrostatic
interactions were calculated using the particle-mesh Ewald method [51]. A smoothing function was
applied to truncate short-range electrostatic interactions. The Verlet-I/r-RESPA multiple time-step
propagator was used to integrate the equation of motions using a time step of 2 and 4 fs for short- and
long-range forces, respectively [52]. All bonds involving hydrogen atoms were constrained using the
Rattle algorithm [53]. The most abundant conformations of the ligand were determined by clustering
one trajectory of the systems 1a and 1b using the G_CLUSTER tool from the MD suite GROMACS [54],
based on the root-mean-square deviation matrix of the ligand.
Supplementary Materials: Supplementary materials are available online. For detailed descriptions and results
on synthetic protocols of the compounds and their NMR structural analyses and the measurements of the potency
of compounds using inhibition assays based on whole cell interactions (HAI) vs. based on molecular interactions
either via competition (SPR) or direct (ITC), see SI.
Acknowledgments: Financial support by the FRQ-NT and MDEIE Québec—Flandres (FWO G.A060.10N)
bilateral program to R.R. and J.B. and of the French “Agence Nationale de la Recherche” (ANR Blanc STARLET,
ANR-12-BSV5-0016-01) to E.M., N.Y. and J.B., is gratefully acknowledged. E.-M.K. has received funding from
the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie
grant agreement No. 750280. R.R. is thankful for the generous financial support from the Natural Sciences and
Engineering Research Council of Canada (NSERC) through a Canadian Research Chair (CRC) in Therapeutic
Chemistry. IC10 were Fab fragments of a monoclonal antibody generated against the FimC (chaperone)-FimH
(adhesin) complex at Medimmune Inc., Gaithersbourg, MD, USA. The 900 MHz NMR spectrometer was funded
by Région Nord-Pas de Calais, the European Union (FEDER), the French Ministery for Research, the Université
Lille 1 and CNRS. We thank the FR3688 federation for use of the TGE RMN THC, and Alexandre Arnold (UQAM)
for expert NMR suggestions and discussions.
Author Contributions: R.R. and J.B. conceived and designed the experiments; M.T., T.C.S., Q.W. and L.M.
synthesized the chemical compounds; J.B. and N.Y. made and interpreted SPR and HAI affinity measurements;
E.-M.K. and M.F.L. performed MD simulations and N.Y. and G.V. did docking experiments; M.T., T.C.S., A.P. and
E.M. collected and analyzed NMR spectra; A.G. and S.O. contributed reagents for the SPR analysis; M.T., E.-M.K.,
R.R. and J.B. finalized the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Kau, A.L.; Hunstad, D.A.; Hultgren, S.J. Interaction of uropathogenic Escherichia coli with host uroepithelium.
Curr. Opin. Microbiol. 2005, 8, 54–59. [CrossRef] [PubMed]
2.
Carvalho, F.A.; Barnich, N.; Sivignon, A.; Darcha, C.; Chan, C.H.; Stanners, C.P.; Darfeuille-Michaud, A.
Crohn’s disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic
mice expressing human ceacam. J. Exp. Med. 2009, 206, 2179–2189. [CrossRef] [PubMed]
3.
4.
Firon, N.; Ofek, I.; Sharon, N. Carbohydrate specificity of the surface lectins of Escherichia coli, Klebsiella
pneumoniae and Salmonella typhimurium. Carbohydr. Res. 1983, 120, 235–249. [CrossRef]
Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools, L.; Wuhrer, M.; Hung, C.S.; Pinkner, J.;
Slattegard, R.; Zavialov, A.; et al. Receptor binding studies disclose a novel class of high-affinity inhibitors of
the Escherichia coli FimH adhesin. Mol. Microbiol. 2005, 55, 441–455. [CrossRef] [PubMed]
5.
6.
Wellens, A.; Garofalo, C.; Nguyen, H.; van Gerven, N.; Slattegard, R.; Hernalsteens, J.P.; Wyns, L.; Oscarson, S.;
de Greve, H.; Hultgren, S.; et al. Intervening with urinary tract infections using anti-adhesives based on the
crystal structure of the Fimh-oligomannose-3 complex. PLoS ONE 2008, 3, e2040. [CrossRef]
Klein, T.; Abgottspon, D.; Wittwer, M.; Rabbani, S.; Herold, J.; Jiang, X.; Kleeb, S.; Luthi, C.; Scharenberg, M.;
Bezencon, J.; et al. Fimh antagonists for the oral treatment of urinary tract infections: From design and
synthesis to in vitro and in vivo evaluation. J. Med. Chem. 2010, 53, 8627–8641. [CrossRef] [PubMed]

Molecules 2017, 22, 1101
16 of 18
7.
8.
9.
Han, Z.; Pinkner, J.S.; Ford, B.; Obermann, R.; Nolan, W.; Wildman, S.A.; Hobbs, D.; Ellenberger, T.;
Cusumano, C.K.; Hultgren, S.J.; et al. Structure-based drug design and optimization of mannoside bacterial
FimH antagonists. J. Med. Chem. 2010, 53, 4779–4792. [CrossRef] [PubMed]
Schwardt, O.; Rabbani, S.; Hartmann, M.; Abgottspon, D.; Wittwer, M.; Kleeb, S.; Zalewski, A.; Smiesko, M.;
Cutting, B.; Ernst, B. Design, synthesis and biological evaluation of mannosyl triazoles as FimH antagonists.
Bioorg. Med. Chem. 2011, 19, 6454–6473. [CrossRef] [PubMed]
Wellens, A.; Lahmann, M.; Touaibia, M.; Vaucher, J.; Oscarson, S.; Roy, R.; Remaut, H.; Bouckaert, J.
The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH.
Biochemistry 2012, 51, 4790–4799. [CrossRef] [PubMed]
10. Jiang, X.; Abgottspon, D.; Kleeb, S.; Rabbani, S.; Scharenberg, M.; Wittwer, M.; Haug, M.; Schwardt, O.;
Ernst, B. Antiadhesion therapy for urinary tract infections-a balanced pk/pd profile proved to be key for
success. J. Med. Chem. 2012, 55, 4700–4713. [CrossRef] [PubMed]
11. Han, Z.; Pinkner, J.S.; Ford, B.; Chorell, E.; Crowley, J.M.; Cusumano, C.K.; Campbell, S.; Henderson, J.P.;
Hultgren, S.J.; Janetka, J.W. Lead optimization studies on FimH antagonists: Discovery of potent and orally
bioavailable ortho-substituted biphenyl mannosides. J. Med. Chem. 2012, 55, 3945–3959. [CrossRef] [PubMed]
12. Brument, S.; Sivignon, A.; Dumych, T.I.; Moreau, N.; Roos, G.; Guerardel, Y.; Chalopin, T.; Deniaud, D.;
Bilyy, R.O.; Darfeuille-Michaud, A.; et al. Thiazolylaminomannosides as potent anti-adhesives of type 1
piliated Escherichia coli isolated from Crohn’s disease patients. J. Med. Chem. 2013, 56, 5395–5406. [CrossRef]
[PubMed]
13. Roos, G.; Wellens, A.; Touaibia, M.; Yamakawa, N.; Geerlings, P.; Roy, R.; Wyns, L.; Bouckaert, J. Validation of
reactivity descriptors to assess the aromatic stacking within the tyrosine gate of FimH. ACS Med. Chem. Lett.
2013, 4, 1085–1090. [CrossRef] [PubMed]
14. Vanwetswinkel, S.; Volkov, A.N.; Sterckx, Y.G.; Garcia-Pino, A.; Buts, L.; Vranken, W.F.; Bouckaert, J.;
Roy, R.; Wyns, L.; van Nuland, N.A. Study of the structural and dynamic effects in the FimH adhesin upon
alpha-d-heptyl mannose binding. J. Med. Chem. 2014, 57, 1416–1427. [CrossRef] [PubMed]
15. Gouin, S.G.; Roos, G.; Bouckaert, J. Discovery and application of FimH antagonists. Top. Med. Chem. 2014
12, 123–168.
,
16. Chalopin, T.; Brissonnet, Y.; Sivignon, A.; Deniaud, D.; Cremet, L.; Barnich, N.; Bouckaert, J.; Gouin, S.G.
Inhibition profiles of mono- and polyvalent FimH antagonists against 10 different Escherichia coli strains.
Org. Biomol. Chem. 2015, 13, 11369–11375. [CrossRef] [PubMed]
17. Sivignon, A.; Yan, X.; Alvarez Dorta, D.; Bonnet, R.; Bouckaert, J.; Fleury, E.; Bernard, J.; Gouin, S.G.;
Darfeuille-Michaud, A.; Barnich, N. Development of heptylmannoside-based glycoconjugate antiadhesive
compounds against adherent-invasive Escherichia coli bacteria associated with Crohn’s disease. mBio 2015
6, e01298-15. [CrossRef] [PubMed]
,
18. Mydock-McGrane, L.; Cusumano, Z.; Han, Z.; Binkley, J.; Kostakioti, M.; Hannan, T.; Pinkner, J.S.; Klein, R.;
Kalas, V.; Crowley, J.; et al. Antivirulence C-mannosides as antibiotic-sparing, oral therapeutics for urinary
tract infections. J. Med. Chem. 2016, 59, 9390–9408. [CrossRef] [PubMed]
19. Mydock-McGrane, L.K.; Cusumano, Z.T.; Janetka, J.W. Mannose-derived FimH antagonists: A promising
anti-virulence therapeutic strategy for urinary tract infections and Crohn’s disease. Expert Opin. Ther. Pat.
2016, 26, 175–197. [CrossRef] [PubMed]
20. Jarvis, C.; Han, Z.; Kalas, V.; Klein, R.; Pinkner, J.S.; Ford, B.; Binkley, J.; Cusumano, C.K.; Cusumano, Z.;
Mydock-McGrane, L.; et al. Antivirulence isoquinolone mannosides: Optimization of the biaryl aglycone for
FimH lectin binding affinity and efficacy in the treatment of chronic UTI. ChemMedChem 2016, 11, 367–373.
[CrossRef] [PubMed]
21. Chalopin, T.; Alvarez Dorta, D.; Sivignon, A.; Caudan, M.; Dumych, T.I.; Bilyy, R.O.; Deniaud, D.; Barnich, N.;
Bouckaert, J.; Gouin, S.G. Second generation of thiazolylmannosides, FimH antagonists for E. coli-induced
Crohn’s disease. Org. Biomol. Chem. 2016, 14, 3913–3925. [CrossRef] [PubMed]
22. Alvarez Dorta, D.; Sivignon, A.; Chalopin, T.; Dumych, T.I.; Roos, G.; Bilyy, R.O.; Deniaud, D.; Krammer, E.M.;
de Ruyck, J.; Lensink, M.F.; et al. The antiadhesive strategy in crohn’s disease: Orally active mannosides to
decolonize pathogenic Escherichia coli from the gut. Chembiochem 2016, 17, 936–952. [CrossRef] [PubMed]
23. Kleeb, S.; Jiang, X.; Frei, P.; Sigl, A.; Bezencon, J.; Bamberger, K.; Schwardt, O.; Ernst, B. Fimh antagonists:
Phosphate prodrugs improve oral bioavailability. J. Med. Chem. 2016, 59, 3163–3182. [CrossRef] [PubMed]

Molecules 2017, 22, 1101
17 of 18
24. Rabbani, S.; Krammer, E.-M.; Roos, G.; Zalewski, A.; Preston, R.; Eid, S.; Zihlmann, P.; Prevost, M.;
Lensink, M.F.; Thompson, A.; et al. Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH
relaxes the tyrosine gate prior to mannose binding. IUCrJ 2017, 4, 7–23. [CrossRef] [PubMed]
25. Hung, C.S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.; DeFusco, A.; Auguste, C.G.; Strouse, R.;
Langermann, S.; Waksman, G.; et al. Structural basis of tropism of Escherichia coli to the bladder during
urinary tract infection. Mol. Microbiol. 2002, 44, 903–915.
26. Dreux, N.; Denizot, J.; Martinez-Medina, M.; Mellmann, A.; Billig, M.; Kisiela, D.; Chattopadhyay, S.;
Sokurenko, E.; Neut, C.; Gower-Rousseau, C.; et al. Point mutations in FimH adhesin of Crohn’s disease-associated
adherent-invasive Escherichia coli enhance intestinal inflammatory response. PLoS Pathog. 2013, 9, e1003141.
[CrossRef] [PubMed]
27. Asensio, J.L.; Arda, A.; Canada, F.J.; Jimenez-Barbero, J. Carbohydrate-aromatic interactions 11. Acc. Chem.
Res. 2013, 46, 946–954. [CrossRef] [PubMed]
28. Fiege, B.; Rabbani, S.; Preston, R.C.; Jakob, R.P.; Zihlmann, P.; Schwardt, O.; Jiang, X.; Maier, T.; Ernst, B.
The tyrosine gate of the bacterial lectin FimH: A conformational analysis by NMR spectroscopy and X-ray
crystallography. Chembiochem 2015, 16, 1235–1246. [CrossRef] [PubMed]
29. Alvarez Dorta, D.; Chalopin, T.; Sivignon, A.; De Ruyck, J.; Dumych, T.; Bilyy, R.; Deniaud, D.; Barnich, N.;
Bouckaert, J.; Gouin, S.G. Physiochemical tuning of potent E. coli antiadhesives by microencapsulation and
methylene homologation. ChemMedChem 2017, 12, 986–998. [CrossRef] [PubMed]
30. Ladeveze, S.; Cioci, G.; Roblin, P.; Mourey, L.; Tranier, S.; Potocki-Veronese, G. Structural bases for N-glycan
processing by mannoside phosphorylase. Acta Crystallogr. D Biol. Crystallogr. 2015, 71, 1335–1346. [CrossRef]
[PubMed]
31. Bertozzi, C.; Bednarski, M.D. Modern methods in carbohydrate synthesis. In Synthesis of C-Glycosides: Stable
Mimics of O-Glycosidic Linkages; Harwood Academic Publishers: Reading, UK, 1996; pp. 316–351.
32. Pang, L.; Kleeb, S.; Lemme, K.; Rabbani, S.; Scharenberg, M.; Zalewski, A.; Schadler, F.; Schwardt, O.;
Ernst, B. Fimh antagonists: Structure-activity and structure-property relationships for biphenyl
α-D-mannopyranosides. ChemMedChem 2012, 7, 1404–1422. [CrossRef] [PubMed]
33. Bouckaert, J.; Mackenzie, J.; de Paz, J.L.; Chipwaza, B.; Choudhury, D.; Zavialov, A.; Mannerstedt, K.;
Anderson, J.; Pierard, D.; Wyns, L.; et al. The affinity of the Fimh fimbrial adhesin is receptor-driven and
quasi-independent of Escherichia coli pathotypes. Mol. Microbiol. 2006, 61, 1556–1568. [CrossRef] [PubMed]
34. Durka, M.; Buffet, K.; Iehl, J.; Holler, M.; Nierengarten, J.F.; Taganna, J.; Bouckaert, J.; Vincent, S.P.
The functional valency of dodecamannosylated fullerenes with Escherichia coli Fimh-towards novel bacterial
anti-adhesives. Chem. Commun.(Camb.) 2011, 47, 1321–1323. [CrossRef] [PubMed]
35. Scharenberg, M.; Schwardt, O.; Rabbani, S.; Ernst, B. Target selectivity of FimH antagonists. J. Med. Chem.
2012, 55, 9810–9816. [CrossRef] [PubMed]
36. Le Trong, I.; Aprikian, P.; Kidd, B.A.; Forero-Shelton, M.; Tchesnokova, V.; Rajagopal, P.; Rodriguez, V.;
Interlandi, G.; Klevit, R.; Vogel, V.; et al. Structural basis for mechanical force regulation of the adhesin FimH
via finger trap-like beta sheet twisting. Cell 2010, 141, 645–655. [CrossRef] [PubMed]
37. Le Trong, I.; Aprikian, P.; Kidd, B.A.; Thomas, W.E.; Sokurenko, E.V.; Stenkamp, R.E. Donor strand exchange
and conformational changes during E. coli fimbrial formation. J. Struct. Biol. 2010, 172, 380–388. [CrossRef]
[PubMed]
38. De Ruyck, J.; Lensink, M.F.; Bouckaert, J. Structures of C-mannosylated anti-adhesives bound to the type 1
fimbrial FimH adhesin. IUCrJ 2016, 3, 163–167. [CrossRef] [PubMed]
39. Tomasic, T.; Rabbani, S.; Gobec, M.; Rašcˇan, I.M.; Podlipnik, C.; Ernst, B.; Anderluh, M. Branched
α
-D-mannopyranosides: A new class of potent FimH antagonists. MedChemComm 2014, 5, 1247–1253.
[CrossRef]
40. Eris, D.; Preston, R.C.; Scharenberg, M.; Hulliger, F.; Abgottspon, D.; Pang, L.; Jiang, X.; Schwardt, O.; Ernst, B.
The conformational variability of FimH: Which conformation represents the therapeutic target? Chembiochem
2016, 17, 1012–1020. [CrossRef] [PubMed]
41. Mikkelsen, L.M.; Hernaiz, M.J.; Martin-Pastor, M.; Skrydstrup, T.; Jimenez-Barbero, J. Conformation of
glycomimetics in the free and protein-bound state: Structural and binding features of the C-glycosyl
analogue of the core trisaccharide
α-D-man-(1 –> 3)-[α-D-man-(1 –> 6)]-D-man. J. Am. Chem. Soc. 2002, 124,
14940–14951. [CrossRef] [PubMed]

Molecules 2017, 22, 1101
18 of 18
42. Kisiela, D.I.; Avagyan, H.; Friend, D.; Jalan, A.; Gupta, S.; Interlandi, G.; Liu, Y.; Tchesnokova, V.;
Rodriguez, V.B.; Sumida, J.P. Inhibition and reversal of microbial attachment by an antibody with parasteric
activity against the fimh adhesin of uropathogenic E. coli. PLoS Pathog. 2015, 11, e1004857. [CrossRef]
[PubMed]
43. Szunerits, S.; Zagorodko, O.; Cogez, V.; Dumych, T.; Chalopin, T.; Alvarez Dorta, D.; Sivignon, A.; Barnich, N.;
Harduin-Lepers, A.; Larroulet, I.; et al. Differentiation of Crohn’s disease-associated isolates from other
pathogenic Escherichia coli by fimbrial adhesion under shear force. Biology (Basel) 2016, 5, 14. [CrossRef]
[PubMed]
44. Stewart, J.J. Optimization of parameters for semiempirical methods vi: More modifications to the nddo
approximations and re-optimization of parameters. J. Mol. Model. 2013, 19, 1–32. [CrossRef] [PubMed]
45. Storer, J.W.; Giesen, D.J.; Cramer, C.J.; Truhlar, D.G. Class IV charge models: A new semiempirical approach
in quantum chemistry. J. Comput. Aided Mol. Des. 1995, 9, 87–110. [CrossRef] [PubMed]
46. Vergoten, G.; Mazur, I.; Lagant, P.; Michalski, J.C.; Zanetta, J.P. The spasiba force field as an essential tool for
studying the structure and dynamics of saccharides. Biochimie 2003, 85, 65–73. [CrossRef]
47. Lagant, P.; Nolde, D.; Stote, R.; Vergoten, G.; Karplus, M. Increasing normal modes analysis accuracy:
The SPASIBA spectroscopic force field introduced into the CHARMM program. J. Phys. Chem. A 2004, 108,
4019–4029. [CrossRef]
48. Bas, D.C.; Rogers, D.M.; Jensen, J.H. Very fast prediction and rationalization of pKa values for protein-ligand
complexes. Proteins 2008, 73, 765–783. [CrossRef] [PubMed]
49. Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.;
Lopes, P.; Vorobyov, I.; et al. Charmm general force field: A force field for drug-like molecules compatible
with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 2010, 31, 671–690. [CrossRef]
[PubMed]
50. Phillips, J.C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R.D.; Kale, L.;
Schulten, K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781–1802. [CrossRef]
[PubMed]
51. Darden, T.Y.D.; Pedersen, L. Particle mesh ewald:An n*log(n) method for Ewald sums in large systems.
J. Chem. Phys. 1993, 98, 10089–10092. [CrossRef]
52. Tuckerman, M.B.; Berne, B.J.; Martyna, G.J. Reversible multiple time scale molecular dynamics. J. Chem.
Phys. 1992, 97, 1990. [CrossRef]
53. Andersen, H.C. Rattle: A “velocity” version of the shake algorithm for molecular dynamics calculations.
J. Comput. Phys. 1983, 52, 24–34. [CrossRef]
54. Pronk, S.; Pall, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.R.; Smith, J.C.; Kasson, P.M.;
van der Spoel, D.; et al. Gromacs 4.5: A high-throughput and highly parallel open source molecular
simulation toolkit. Bioinformatics 2013, 29, 845–854. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
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