Hexavalent thiofucosides to probe the role of theAspergillus fumigatuslectin FleA in fungal pathogenicity

Article abstract of DOI:10.1039/d1ob00152c

Aspergillus fumigatusis a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role inA. fumigatuspathogenesis was recently identif

Full text of DOI:10.1039/d1ob00152c

Organic &
Biomolecular Chemistry
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Hexavalent thiofucosides to probe the role of the
Aspergillus fumigatus lectin FleA in fungal
pathogenicity†
Cite this: Org. Biomol. Chem., 2021,
19, 3234
Christophe Dussouy,a Pierre-Alban Lalys,a Aurore Cabanettes,b Victor Lehot,a
David Deniaud, a Emilie Gillon,b Viviane Balloy,c Annabelle Varrot *b and
a
Sébastien G. Gouin
*
Aspergillus fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety
of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in
A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic
stability are needed to explore the role of FleA in the infection process. In this study, we developed potent
FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a
set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The
most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation.
Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly
enhanced binding affinity (Kd = 223 21 nM) thanks to a chelate binding mode. The compound could
probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasion.
Received 26th January 2021,
Accepted 15th March 2021
DOI: 10.1039/d1ob00152c
rsc.li/obc
pies based on antiadhesive molecules. Such a strategy is less
prone to antimicrobial resistance than existing treatments as
Introduction
Aspergillus fumigatus is one of the most ubiquitous saprophytic selective pressure is not exerted on the microorganisms in the
fungi, with an ecological niche in the soil. A. fumigatus abun- same way. This promising antiadhesive strategy has been par-
dantly disseminates conidia into the atmosphere, which are ticularly explored in recent decades,2–4 with several studies
easily inhaled into the lungs where they reach the pulmonary reporting significant in vivo effects. Striking decolonization
alveoli due to their small size. Immunocompetent hosts efficien- and antibiofilm effects have been reported on bacterial lectins
tly clear the conidia out from their respiratory system by such as the Shiga-like and cholera toxins,5,6 FimH expressed
mechanical mucus elimination or innate immunity. However, by pathogenic Escherichia coli strains implicated in urinary
A. fumigatus is a severe opportunistic pathogen for immunocom- tract infections7–9 or inflammatory bowel diseases,10–12 LecA
promised patients in whom may cause generally fatal lung infec- and LecB expressed by Pseudomonas aeruginosa in the context
tions such as invasive aspergillosis or allergic bronchopulmon- of lung infections.13–15 This promising concept demonstrated
ary aspergillosis.1 Increasing resistances to antifungal drug on bacteria, has now reached the clinical stage, and may be
treatments such as the azole class of antifungal agents has fos- potentially applied to other classes of pathogens such as fungi.
tered research to identify the virulence factors of A. fumigatus.
Recently, Armstrong and coll. identified a lectin named FleA
Carbohydrate-binding proteins (lectins) from microbial (or AFL) from A. fumigatus which shares homologies with a
pathogens are one of the most common group of receptors fucose-binding lectin from the orange peel mushroom Aleuria
involved in the primary steps of cell infection. Lectins aurianta.16 The role of the lectin is still unclear and it was
expressed by bacteria or fungi and mediating host adhesion initially suggested to help the fungus to thrive in decaying
are therefore promising targets for the development of thera- matter, to be an important virulence factor involved in the
early stage of colonization and to contribute to the inflamma-
tory response.17 However, studies have also shown that FleA
aUniversité de Nantes, CNRS, CEISAM UMR, 6230, F-44000 Nantes, France.
E-mail: sebastien.gouin@univ-nantes.fr
bUniv. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
cSorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, Paris,
France
recognition by bronchial epithelial cells attenuates conidial
germination,18 and that mucin binding and macrophage
killing may prevent A. fumigatus pneumonia.19 Thus, there is a
strong interest in developing molecular antagonists of FleA to
fully probe the role of this lectin in the fungal pathogenicity
and to assess antagonist therapeutic potential.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00152c
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X-ray diffraction of FleA co-crystallized with methyl-α-L-sele- synthesis of the monovalent α-O-fucosides 1, 3, 4 and 6
nofucoside gave structural insights into this first characterized (Scheme 1). Sulfuric acid immobilized on silica24 catalysed
lectin from pathogenic fungi.17 The lectin forms homodimers α-addition of propargyl alcohols (synthesis in ESI†) to fucose,
of six-bladed β-propellers. Although the six sugar binding sites leading to the expected compounds 15–18 after acetate protec-
(one per blade) are non-equivalent and have specific oligosac- tion. The fucosides were engaged in a copper-catalysed azide–
charide preferences, FleA shows a marked preference for alkyne cyclization in the presence of sodium ascorbate to form
α-anomeric fucosides.20 We and others have exploited these exclusively 1,4-regioisomers 19–22 as shown by the large
specific features to design synthetic multivalent fucosides with Δ(δc-4–δc-5) observed by 13C NMR.25
the potential ability to interact simultaneously in several
binding sites of FleA by a chelate binding mode (Fig. 1A).21–23
In particular, we recently showed that hexavalent fucosides
based on a cyclodextrin (CD) core and optimized oligo(ethyle-
neglycol) spacers (Fig. 1B) are potent FleA antagonists and can
decrease spore adhesion to pneumocytes at low micromolar
concentrations.21 In the present work, we designed a second
generation of hexavalent fucosides. A small library of mono-
valent fucosides 1–10 was first designed (Fig. 1C, Step 1) and
their binding affinity for FleA assessed by isothermal microca-
lorimetry (ITC) (Step 2). The most promising monovalent ligand
could be cocrystallized in complex with FleA and was selected to
design the corresponding hexavalent fucoside (Step 3).
Results and discussion
FleA displays a slight preference for the α-fucosides compared
with the corresponding β-anomers.20 Thus, a Fischer glycosyla-
Scheme 1 Chemical synthesis of the monovalent α-O-fucosides 1, 3,
4, 6.
tion protocol was employed in the first step of the chemical
Fig. 1 (A) Multivalent fucosides binding the FleA target in a chelate fashion. (B) In a previous study, simple fucose sugar was appended to a cyclo-
dextrin core.21 (C) In the present study, we designed a new generation of fucosides with varied hydrophobic groups at the anomeric position. A
three-step process was used to obtain optimized and non-hydrolysable hexavalent fucosides.
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Excess copper was removed from the crude product after
Organic & Biomolecular Chemistry
Table 1 Binding affinity of compounds 1–10 towards FleA as deter-
mined by ITC
overnight stirring with a chelex resin prior to chromatography
on silica gel. The acetate groups were removed using a basic
resin to form the monovalent α-O-fucosides 1, 3, 4 and 6.
Thiofucosides 2, 5 and 7–10 were obtained from the
common intermediate 1-thio-α-L-fucopyranose tetraacetate
2326 (Scheme 2). The thioacetate group was first deprotected as
a thiolate 24 using potassium carbonate and sodium metha-
nethiolate in methanol. Surprisingly, using a mixture of the
two bases proved very efficient at forming 24. The thiolate was
directly engaged in a thiol–ene click reaction with commer-
cially available vinylic synthon 29 and 30 to form 31 and 32
with 57 and 50% yields, respectively. Then, the aromatic
hydroxyl groups were alkylated with propargyl bromide 25 in
presence of potassium carbonate to form 37 and 38.
Compounds 33–36 were also obtained from the crude thiolate
24, directly engaged in a nucleophilic substitution reaction of
bromo- or tosylated alkynyl-armed synthons 25–28 (synthesis
in ESI†). The CuAAc protocol to form 19–22 was repeated with
33–38 and led to good yields of pure cycloadducts 39–44 and
the expected thio-fucosides 2, 5, 7–10 after acetates hydrolysis
with IRN78.
Cpd
R=
Kd (µM)
110
55.8 1.8
48.8 4.1
53.8 3.3
59.5 0.0
100.7 0.7
46.7 1.9
β
MF
1
2
—
2
—
2.0
2.2
2.0
1.8
1.1
2.4
3
4
5
6
7
8
90.6 3.7
18.5 0.6
1.2
5.9
9
89.4 3.2
1.2
1.1
10
103.9 4.9
Once we had the monovalent fucosides 1–10 in hand, we
assessed their affinity towards FleA by isothermal titration
calorimetry (ITC). We initially performed docking experiments respectively. This is however not a general rule, as 1 and 2 with
with the monovalent fucosides. Unfortunatly, the results (not O-CH2 and S-CH2 R groups displayed virtually identical affinity
shown) poorly correlated with ITC ranking. The theoretical pre- constants. The impact of methylene homologation was shown
dictive efficiency of the compounds is challenging on the FleA to be insignificant in the aliphatic series of compounds 1, 3
target possessing six non-equivalent fucose binding pockets. and 4 but dramatically reduced FleA affinity when comparing
Dissociations constants Kd and relative affinity values (β = 8 and 9 with phenyl rings. Thus, subtly different interactions
KdMF/KdCpd) towards α-methylfucoside (MF, Kd = 110 µM)21 are operate depending on the anomeric substituents and a larger
presented Table 1. All of the compounds showed improved library of fucosides may allow the identification of more
potency compared with MF, with β values ranging to 1.1 to 5.9. potent submicromolar FleA antagonists.
Switching the anomeric oxygen for a sulfur atom was detri-
Ligand 8 (Kd = 18 µM), showed a significantly higher affinity
mental with thio-compounds 5 and 7 showing around two fold for FleA compared with MF or compounds 1–7, 9 and 10. In
higher Kd values compared with the O-analogues 4 and 6, consequence, we decided to study its binding mode with FleA
in greater depth by X-ray crystallography. 8 is bound to five out
of the six binding sites of FleA (Fig. 2). In site 4, we find a gly-
cerol or a PEG molecule bound, since it is more deeply buried.
The binding of compound 8 will be inhibited here by steric
clashes of the aglycone moiety with the protein and in particu-
lar with the side chain of Tyr168. In molecule B, a glycerol
moiety is also observed in site 6 that could result in binding
difficulties due to the crystal contacts. In site 5, only electron
density for the fucosyl moiety is visible. In sites 1–3, the
phenyl moiety of ligand 8 could be modelled in the electron
density. In site 1 of molecule A, we observed electron density
up to the triazole ring of compound 8 (Fig. 2D, section 1A). No
electron density was visible for the oligo(ethylene glycol)
spacer as it is exposed to the solvent which leads to disorder.
The interactions with the fucose moiety are the same as pre-
viously described.20 The phenyl moiety does not interact
directly with the protein but is visible in sites 1–3 where some
hydrophobic interactions would stabilize its conformation. In
site 1, the aglycone displays two conformations: one where it
Scheme 2 Chemical synthesis of the monovalent α-S-fucosides.
makes hydrophobic contact with Leu39 and the side chain of
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Fig. 2 Crystal structure of FleA in complex with ligand 8. (A) Representation of FleA dimer coloured by protomer. (B) FleA beta propeller coloured
by blade represented in cartoon representation for protomer B, and (C) in surface representation for protomer A. (D) Zoom on FleA binding sites
with 2Fo-DFc electron density represented at 1 σ (0.4 eÅ3). Panels are labelled by site and protomer. Ligands are represented in balls and sticks.
Glu41 and one where the triazole ring stacks against the the highest FleA affinity of the thiofucosides serie. Several
indole ring of His23 (Fig. 2B).
scaffolds may be used for the efficient design of multivalent
Based on the ITC results obtained with monovalent deriva- fucosides.22 αCD scaffolds are good candidates due to their
tives 1–10, we decided to synthesize the CD fucosides 48 and hydrophilic nature and rigid core limiting nonspecific inter-
49, which are hexavalent clusters of ligands 2 and 8 displaying actions with proteins and entropic penalty upon ligand
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Table 2 Binding affinity of compounds 48 and 49 towards FleA as
determined by ITC
binding. They are easily functionalized at the primary rim to
present the sugar ligands with an angular spatial restriction.27
In a previous study,21 we showed that azido-αCD analogues
could be easily functionalized by simple alkyne O-fucosides
using click chemistry techniques to form water-soluble hexa-
valent CD. We also optimized the spacer arm length, and the
best affinity for FleA was obtained with a tetraethyleneglycol
spacer. For these reasons, functionalized scaffold 45 was
selected in the present study. Only S-fucosides were clusterized
due to their potential inertness to glycosidase hydrolysis. The
ligands were grafted by CuAAc on azido-functionalized CD 45,
obtained by a previously described protocole.9 Acetate de-
protection on a basic resin followed by purification on a
Sephadex column led to the expected hexavalent compounds
48 and 49 (Scheme 3).
Next, the binding affinity of 48 and 49 for FleA was assessed
by ITC and compared to MF. In this assay, the reference com-
pound MF showed a similar dissociation constant value as pre-
viously published21 (Kd = 98 vs. 110 μM). Surprisingly, the
differences in binding affinity of monovalent 2 and 8 for FleA
disappeared when the compounds were multimerized on CD.
Indeed, hexavalent derivatives 48 and 49 showed rather similar
Kds measured by ITC (Table 2). The fact that the relative inhibi-
tory potency of the fucosides is not conserved when 2 and 8
are multimerized may be related to the non-equivalence of the
six binding sites of FleA. Nevertheless, the hexavalent com-
pounds were much more potent than their monovalent ana-
logues, with Kds reaching the nanomolar inhibition level. Each
clustered fucoside was more than 400-fold more potent com-
pared to MF. This sygnergistic effect may be explained by a
partial chelation of the FleA binding sites as suggested by the
lower than 1 average stoechiometry (n) values obtained for 48
Cpd
R=
Kd (nM)
β
MF
48
49
—
98 200
238.5 28.5
223 21
0
—
412
440
(n = 0.814 0.155) and 49 (n = 0.784 0.006) from three inde-
pendant ITC measurements.
Next we evaluated the potency of 49 to probe the respective
role of FleA in A. fumigatus pathogenesis. In a previous study,
Balloy and co-workers investigated the mechanisms of
A. fumigatus conidia clearance from the lung.18 Human
bronchoepithelial cells were shown to inhibit the filament for-
mation of extracellular A. fumigatus (conidia germination into
hyphae) via FleA recognition. FleA from conidia bound at cell
surfaces led to cell receptors clustering with enhanced avidity,
triggering a fungistatic process that involves the phosphoinosi-
tide 3-kinase pathway. This antifungal activity could be abol-
ished by cell-receptors saturation, with recombinant FleA.
Here, we assessed if a similar effect could occur after specific
binding of 49 to FleA from conidia. The level of conidia germi-
nation into hyphae (filamentation) was followed by the release
of galactomannan in the medium (Fig. 3). Pre-incubation of
BEAS-2B epithelial cells with FleA (1 µM) before (1 hour) and
during infection (15 hours) with A. fumigatus conidia led to a
much higher level of filamentation, as previously reported,18
and shown by a higher galactomannan level (Fig. 3, Af + FleA).
Interestingly, a similar trend was observed when 49 (100 µM),
instead of FleA, was co-incubated with the conidia and the
cells (Af + 49). This inhibition of the fungistatic process was
however not observed with the less potent FleA antagonist MF
at 100 µM (Af + MF). Thus, these preliminary results suggest
that optimized multivalent fucosides such as 49 are valuable
probes to study A. fumigatus pathogenesis.
Fig. 3 Effect of FleA inhibition on galactomannan release. Values,
expressed in optical density (O.D.), are presented as mean SEM ****p <
Scheme 3 Chemical synthesis of the hexavalent α-S-fucosides 48 and
49.
0.0001, *p
Bonferroni’s multiple comparison test).
< 0.05, ns: not significant (ANOVA test, followed by
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Conclusions
Fucose-binding lectins expressed by life-threatening pathogens
such as B. cenocepacia or A. fumigatus have attracted attention
in view of their potential implications in respiratory
infections.19,21,22,28,29 The role of the recently identified FleA
lectin in A. fumigatus pathogenesis is however still unclear.
Published studies suggest that FleA-promoted binding may
actually attenuates A. fumigatus virulence, by improving muco-
cilliary clearance, macrophage killing, and/or inhibition of
conidia germination to hyphae.18,19 Thus new chemical probes
are required to better understand the complex role(s) of FleA
during A. fumigatus infections. In this work, we developed non-
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Author contributions
Conceptualization, supervision and writing: S.G. and A.V. revis-
ing: D. D. acquisition and formal analysis: C.D.; P-A. L. and V.
L. (chemistry), A.V. crystallography, A.C. and E.G. (ITC), V.B.
(cell assay).
Conflicts of interest
14 R. Sommer, K. Rox, S. Wagner, D. Hauck, S. S. Henrikus,
S. Newsad, T. Arnold, T. Ryckmans, M. Brönstrup,
A. Imberty, A. Varrot, R. W. Hartmann and A. Titz, J. Med.
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17 J. Houser, J. Komarek, N. Kostlanova, G. Cioci, A. Varrot,
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There are no conflicts of interest to declare.
Acknowledgements
The authors are grateful for access Proxima 1 at SOLEIL
Synchrotron, Saint Aubin, France (proposal number
20170827). This work was supported by the SATT OUEST
VALORISATION, the French National Research Agency
(Glyco@Alps grant no. ANR-15-IDEX-02) and the Centre
National de la Recherche Scientifique (CNRS).
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