Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases

Article abstract of DOI:10.1002/chem.201805790

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Full text of DOI:10.1002/chem.201805790

A Journal of
Accepted Article
Title: Multivalent thiosialosides and their synergistic interaction with
pathogenic sialidases.
Authors: yoan Brissonnet, coralie assailly, Amélie Saumonneau,
Julie Bouckaert, Mike Maillasson, Clémence Petitot, benoit
roubinet, Blanka Didak, Ludovic Landemarre, Clarisse Bridot,
Ralf Blossey, david deniaud, xibo yan, julien bernard, Charles
Tellier, cyrille grandjean, Franck Daligault, and Sébastien G.
Gouin
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201805790
Link to VoR: http://dx.doi.org/10.1002/chem.201805790
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10.1002/chem.201805790
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Multivalent thiosialosides and their synergistic interaction with
pathogenic sialidases.
Yoan Brissonnet,[a] Coralie Assailly,[a] Amélie Saumonneau,[b] Julie Bouckaert,[c] Mike Maillasson,[d]
Clémence Petitot,[b] Benoit Roubinet,[e] Blanka Didak,[e] Ludovic Landemarre,[e] Clarisse Bridot,[c] Ralf
Blossey,[c] David Deniaud,[a] Xibo Yan,[f] Julien Bernard,[f] Charles Tellier,[b] Cyrille Grandjean,[b] Franck
Daligault[b] and Sébastien G. Gouin.[a]*
Abstract: Sialidases (SA) hydrolyze sialyl residues from
glycoconjugates of the eukaryotic cell surface and are virulence
factors expressed by pathogenic bacteria, viruses and parasites. The
catalytic domain of SA are often flanked with carbohydrate-binding
module(s) previously shown to bind sialosides and to enhance
enzymatic catalytic efficiency. Here we designed non-hydrolyzable
multivalent thiosialosides as probes and inhibitors of V. cholerae, T.
cruzi and S. pneumoniae (NanA) sialidases. NanA was truncated from
the catalytic and lectinic domains (NanA-L and NanA-C) to probe their
respective roles when interacting with sialylated surfaces and the
synthetically designed di-, and polymeric thiosialosides. NanA-L
domain was shown to fully drive NanA binding, improving affinity for
the thiosialylated surface and compounds by more than two orders of
magnitude. Importantly, each thiosialoside grafted onto the polymer
was also shown to reduce NanA and NanA-C catalytic activity with a
3000-fold higher efficiency compared to the monovalent thiosialoside
reference. These results extend the concept of multivalency for
designing potent bacterial and parasitic sialidase inhibitors.
Introduction
In the exoglycosidase classes, the sialidases (SA), or
neuraminidases, that occur in animals, virus, pro- and eukaryotic
microorganisms, are of particular interest for developing new anti-
infective therapies. In a wide range of virus and pathogenic
bacteria, SA have been reported as virulence factors removing
the sialic acid from sialoglycan molecules of host cells, in the
primary step of infection. SA activity unmasks underlaying ligands
in which the bacteria and their toxins can adhere and/or be
internalized.[1] Bacterial SA have also been shown to promote the
formation of biofilms, although the exact mechanism has not been
elucidated.[2] Parasites such are Trypanosomes are unable to
synthesize sialic acids but express trans-sialidases that are used
to transfer sialic acids from the host cells to coat glycans on the
pathogen surface. This dampens the host immune system and
helps parasite survival.
Although all exo-sialidases from glycoside hydrolase 33 family
have a common catalytic domain (CAT) with a six-bladed β-
propeller fold, most of them also contain various additional
domains including membrane-binding domains, carbohydrate-
binding modules (CBM), or lectin-like domains (LD). This
structural diversity has been poorly studied and has not yet been
exploited to design efficient and specific SA inhibitors. In a
pionneering study, Boons and co-workers showed that large SA
from C. perfringens or V. cholerae flanked with a lectin domain,
were more efficient in hydrolyzing sialic acid residue from a
polyvalent 3’-sialyl N-acetyllactosamine substrate, than to a
monovalent sialyl analogue.[3] This effect was not observed with
the small SA from S. typhimurium that lack a CBM, highlighting
the crucial role of the lectin domain in SA catalytic efficiency. On
the other hand, previous reports described the efficiency of
glycoclusters[4] bearing multiple sialic acids or transition state
sialosyl cations analogues for the synergistic detection[5,6] and
inhibition of sialic acid binding proteins[7,8] or viral
neuraminidase,[9–12] respectively.
[a]
[b]
Dr Y. Brissonnet, C. Assailly, Prof. Dr. D. Deniaud, Dr. S.G. Gouin
Université de Nantes, CEISAM, Chimie Et Interdisciplinarité,
Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR des
Sciences et des Techniques, 2 rue de la Houssinière, BP 92208,
44322 Nantes Cedex 3, France
E-mail: sebastien.gouin@univ-nantes.fr
Dr. A. Saumonneau, C. Petitot, Prof. Dr. C. Tellier, Dr. C.
Grandjean, Dr. F. Daligault
Université de Nantes, UFIP, UMR CNRS 6286, UFR des Sciences
et des Techniques, 2 rue de la Houssinière, BP 92208, 44322
Nantes Cedex 3, France.
[c]
[d]
[e]
[f]
Dr. J. Bouckaert, Dr. C. Bridot, Dr R. Blossey
Unité de Glycobiologie Structurale et Fonctionnelle (UGSF),
UMR8576 CNRS, Université de Lille 1, Lille 59000, France
Dr. M. Maillasson
Impact biogeneouest, CRCINA, Inserm, CNRS, Université de
Nantes, Nantes, France.
Dr. B. Didak, Dr L. Landemarre
Glycodiag, Bâtiment Physique-Chimie, Rue de Chartres, BP6759,
45067 Orléans cedex 2, France
Inspired by these results, we reasoned that multivalent
thiosialosides could act as potent inhibitors of pathogenic SA by
binding the CAT and/or CBM domains. Thiosialosides should be
stable to enzymatic hydrolysis thanks to the anomeric sulfur atom,
and able to interact in the CAT and CBM when multivalently
displayed on an appropriate scaffold and linker. Furthermore
these glycoclusters are interesting probes to study the
contribution of the CAT and CBM domains in the SA catalytic
efficiency in more depth.
Dr. X. Yann, Dr J. Bernard
Université de Lyon, Lyon, F-69003, France ; INSA-Lyon, IMP,
Villeurbanne, F-69621, France; CNRS, UMR 5223, Ingénierie des
Matériaux Polymères, Villeurbanne, F-69621, France
Supporting information for this article is given via a link at the
end of the document
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Divalent sugars with a fine-tuned spacer arm length have proved
efficient binders for the multimeric cyclic nucleotide-gated
channels[13] and lectin such as wheat germ agglutinin (WGA),[14]
dendritic cell-specific intercellular adhesion molecule-3-grabbing
non-integrin (DC-SIGN),[15] and LecA from the bacteria
Pseudomonas aeruginosa.[16,17] The synergistic effects observed
were ascribed to a highly favorable protein chelation by the
second sugar ligand due to the dramatically increased sugar
concentration in the second binding site proximity. To evaluate
the possibility of reaching such a chelation on the CAT+CBM, we
designed divalent sialosides 2-8 using different sized PEG linkers
(from n=2 to n=130, Figure 1A). The largest length, n=130, is
within 5% of the theoretically maximal multivalent effect according
to the statistical physics model which assumes a Gaussian chain
for polymer (Figure S1).[18] The effective length of the PEG
spacers in solution is much shorter than the extended
conformation and proportional to the root mean square of the PEG
molecular weight.[13] Using this polymer theory we empirically
estimated the average sugar distance for 2-7 which varies from
12 to 67Å.
A)
B)
Figure 1. A) Structure of the synthetic thiosialosides 1-9. B) Structure of the SA targets where the catalytic domain (in red) is flanked with a lectinic domain (in
green).
Polymeric sugars can surpass the inhibitory potency of their
monovalent references by several orders of magnitudes.[19–22]
This was explained by a model where the multimeric lectin
jump from one epitope to another along the polymer chain,
reducing the kinetic off-rate and thus enhancing the affinity.[23]
To estimate if such “bind and jump” mechanism can occur with
the targeted SA, we designed the glycopolymer 9 with an
average of 130 thiosialoside ligands per chain. To compare the
potential improvement in affinity or inhibition due to
multivalency, a monovalent reference is required with chemical
groups in the binding site environment structurally as close as
for the multivalent 2-9. Consequently, we designed
thiosialoside 1 bearing a triazol group and a short PEG spacer.
The enzyme targets selected were three different exo-
sialidases (Vibrio cholerae sialidase- VcSA, Trypanosoma
cruzi trans-sialidase – TcTS, Streptococcus pneumoniae
Neuraminidase A - NanA) which are representative of the
structural variety found in the GH33 family in terms of an
additional carbohydrate binding module flanked to the catalytic
site (Figure 1B). The structural domains of these SA have been
solved and are available from the Protein Data Bank (PDB),[24–
26]
which will be used for interpretation of the inhibition and
binding data.
VcSA removes sialic acid from higher order ganglioside to
reveal GM1 ganglioside, a ligand for the cholera toxin, which
causes acute infection of the intestine. VcSA contains a
catalytic domain flanked by two lectin-like domains (Figure 1B).
For the LD at the C-terminal end of the catalytic domain, the
ligand is unknown, but the other LD at the terminal end binds
to the sialic acid with a Kd of 30 µM, which is one of the highest
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affinity reported for the recognition of a monosaccharide by a
CBM.[24]
unique opportunity to decipher their relative role in sugar
binding and catalytic efficiency.
TcTS from the protozoan catalyze the transfer of sialic acid
residue from host to parasite glycoconjugates, which is critical
for T. cruzi survival and cell invasion capability. T. cruzi is the
causative agent of Chagas’disease, which has affected over
20 million people in South America. TcTS displays a lectin-like
domain (LD) fused to the C-terminal end of the catalytic domain.
Little is known about the function and ligand specificity of these
LDs but glycan array studies indicate that mannose-containing
oligosaccharides and Gal, GalNAc and LacNAc containing
oligosaccharides are binding partners of these LDs.[25]
This work mainly focuses on NanA, a virulence factor present
in 100% of strains from S. pneumoniae,[27,28] causing
meningitis, septicemia and respiratory tract infections. The
catalytic domain of NanA is flanked by a N-terminal CBM40
domain previously shown to be critical in S. pneumoniae
adherence and invasion of the blood-brain-barrier.[29]
To date, inhibitors of NanA were only directed towards the
catalytic domain,[30] but in recent cloning, ligand analysis and
crystallization of the CBM domain revealed a specificity for
sialosides which may open new perspectives in terms of drugs
design.[26,31] Here, the affinity and inhibitory activity of the
synthetic multivalent thiosialosides were assessed against full
length NanA, the catalytic domain (NanA-C) and lectin domain
(NanA-L). The production of the truncated domains offers a
Results and Discussion
Chemical synthesis of the thiosialosides. The chemical
synthesis (Scheme 1) starts with the hydroxyl protection and
anomeric activation of N-acetylneuraminic acid 10 in two steps
to form 11.[32] The anomeric sulfur atom of 12 can be easily
introduced by a nucleophilic substitution of the chlorine atom
with potassium thioacetate in acetone. We failed to obtain 13
by chemoselective thioacetate deprotection protocol with
sodium methanolate at low temperature, followed by
propargylation of the thiol.[33] 13 was obtained from crude 12
after acetate deprotection and thioalkylation with propargyl
bromide. The ligand could be efficiently grafted to scaffold
azides 14-22 (synthesis in SI) by copper-catalyzed azide-
alkyne cyclization (CuAAc) with yields ranging from (43 to
66%) for compounds 23-31. Residual copper was removed
after washing with EDTA or using a Chelex resin. After acetate
deprotection under Zemplèn conditions, the high molecular
weight compounds 3-9 were purified by size-exclusion
chromatography.
ii) NaSMe, MeOH, 0°C, 1h
OAc
i) KSAc, acetone
0°C, 2h30
CO2Me
OAc
OAc
OAc
OAc
Cl
CO2Me
S
AcO
CO2Me
SAc
AcO
AcO
OAc
iii)
Br
ref 29
0°C, 1h
O
O
O
AcHN
10
AcHN
AcHN
AcO
AcO
12
AcO
11
13 (61% for 3 steps)
OAc
OAc
CO2Me
S
CO2Me
S
AcO
AcO
23, 64%
1, 81%
OAc
OAc
iv) A, B or C
v) LiOH
O
O
O
AcHN
N
N
AcHN
OH
2
CuSO4, Na Asc, 60-95°C
N
AcO
AcO
13
24 n = 2, 62%
25 n = 10, 66%
26 n = 30, 62%
27 n = 43,59%
28 n = 66, 56%
29 n = 86, 43%
30 n = 130, 48%
2 n = 2, 91%
R
B
A
3 n = 10, 89%
4 n = 30, 93%
5 n = 43, 91%
6 n = 66, 96%
7 n = 86, 95%
8 n = 130, 96%
O
O
N3
N3
OH
O
N3
v)
O
O
R
n
2
O
R
14
n
15 n = 2
16 n = 10 19 n = 66
17 n = 30 20 n = 86
18 n = 43
C
H
N
O
N3
O
21 n = 130
2
H
N
v)
130
O
31, 54%
9
, 78%
22
R
O
2
O
130
Scheme 1. Chemical synthesis of the sialosides 1-9.
substrate releasing sialic acid and fluorescent 4-
methylumbelliferone (λex = 360 nm and λem = 455 nm) after
enzymatic hydrolysis (Figure 2A). The IC50mol of the compound
is classically reported and defined as the molecule
concentration required to inhibit the initial hydrolysis velocity of
the enzyme by 50%. However, for multivalent ligands this led
Inhibitory activity of the thiosialosides. VcSA is
commercially available, the full procedure to produce TcTS
and the NanA, NanA-C and NanA-L constructs is described in
the supplementary material.
The inhibitory activity of 1-9 against VcSA, TcTS and the NanA
constructs was studied with 2'-(4-methylumbelliferyl)-α-
D-N-
to inflated inhibitory potencies and
a more objective
acetylneuraminic acid (4-MU-NANA) as the enzymatic
comparison of the inhibition requires to correct this value by
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si
the ligand valency. All the IC50 values are reported herein in
mole of sialosides (IC50si) instead of molecules (IC50mol), the
inhibition of TcTS (IC50 = 666 µM). These results confirmed
that a monovalent interaction of simple sialosides with VcSA or
TcTS, lacking the lactose unit of the natural substrates α-2,3-
sialyllactose and α-2,6-sialyllactose is weak. Sialyllactose was
previously crystallized in the catalytic domain of VcSA and a
non-hydrolysable analog was shown to interact strongly ( Kd of
30 µM) with the CBM.[24] For TcTS, lactose displayed a much
greater affinity (Km = 10 µM) compared to sialic acid (Km = 6
mM).[25] The low inhibition observed here was not improved
with divalent 2-8.
si
former being corrected by the valency V (IC50 = V x IC50mol).
The relative inhibitory potency (RIP) compared to our
1
monovalent reference 1 (RIP = IC50 / IC50si) will also be
provided to directly assess the level of a potential synergistic
(or multivalent) effect (if RIP>1).
IC50si values obtained for VcSA, TcTS, NanA and NanA-C after
Dixon plots analysis (Figures S3) are presented in Figure 2B.
The monovalent ligand 1 did not show any inhibition (n.i.) for
VcSA at high concentration (4 mM) and only a moderate
Figure 2. A) Illustration of the enzymatic assay. B) Inhibitory activity of thiosialosides 1-9 against VcSA, TcTS, NanA and NanA-C. IC50 values are expressed in mol
of thiosialosides (valency-corrected). Experiments were performed in triplicate. C) Valency-corrected inhibitory potency of multivalent thiosialosides 2-9 compared
to 1 (RIP values).
If rebinding effects and chelate binding modes are potentially
operating, they do not seem to significantly impact catalytic
activity. In stark contrast, sialosides attached to the polymer 9
proved to be highly potent inhibitors of both SA. Their inhibitory
activity against VcSA or TcTS was improved by at least 39-fold,
and by 208-fold (Figure 2C), respectively. The low IC50si of 3.2
µM with TcTS surpasses previously reported IC50 value for the
sialic acid transition state analogue DANA,[34] and are the best
TcTS inhibitors described so far.[35]
When assessed against NanA and NanA-C, the compounds
showed a highly similar IC50 variation on the full and truncated
domains. RIP of divalent inhibitors 2-8 consistently improved
as the spacer arm length is elongated from n = 2 to n = 130
(Figure 2C). This effect was fully ascribed to the sialoside
ligands as PEG spacers of similar size did not inhibit the
enzymes (results not shown). The maximal RIP and minimal
si
IC50 achieved within the series was 106 and 4 µM when
compound 8 was tested against NanA-C. As previously
observed with VcSA and TcTS, the polymer 9 was by far the
most potent of the series, showing outstanding IC50si values of
343 nM and 143 nM for NanA and NanA-C, respectively. We
subsequently determined the corresponding inhibitory
constants (KiSi) by Dixon and Lineweaver-Burk plots analysis
and found 409 ± 65 nM and 335 ± 40 nM for NanA and NanA-
C with an inhibition profile in accordance with a competitive
binding mode in both cases (Figures S4 and S5). Thus far, this
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represents the best inhibitor for this enzyme and an affinity
improvement per sialosides of around 3000-fold. Surprisingly,
the high affinity improvement observed is therefore not related
to the presence of the lectin domain and 9 showed very similar
RIP value whether tested with NanA-C or NanA (2979 vs 2927).
lectin domain, illustrating its crucial role in anchoring the
sialidases to a sialylated membrane.
Aggregative behavior of NanA-C. We first hypothesized
that a possible multimerization of the NanA-C domains in
solution may compensate the absence of the CBM and
dramatically improve polymer affinity through chelate effects.
Engineered V. cholerae CBM40 into tetramers was previously
shown to improve avidity for a sialic-acid rich surface
compared to the single CBM40 by around three orders of
magnitude.[36] A multimeric NanA-C domain may potentially
provide such synergistic effect and we carefully studied the
oligomerization state of the full and truncated NanA forms by
dynamic light scattering (DLS), small-angle X-ray solution
scattering (SAXS), Multi Angle Light Scattering (MALS), and
size-exclusion chromatography (SEC).
DLS data indicated that solutions of NanA, NanA-C (with initial
concentrations respectively at 19µM and 43µM) have very
similar profiles and contain a major species for NanA and
NanA-C with respectively 83% and 74% of intensity scattered
by the smaller molecular weight (Figures S6 and S7). The three
protein samples were further analyzed by SAXS and MALS
and the experimental molecular weight values found for the
molecular envelopes closely matched the calculated values of
the monomeric constructs (Figures S8 and S9, Table S1).To
estimate the concentration limit at which NanA-C started to
form higher molecular weight structures, different sample
concentrations were analyzed by SEC (Superdex 200pg
16/600, Thermo Scientific®). The unique peak attributed to
NanA (Vr = 84 mL, C=13 µM) was shown to decrease when
the protein concentration was increased to 64 µM (5 mg/ml),
and higher molecular weight structures appeared at Vr = 37.49
mL and Vr = 47 mL (Figure 3).
Figure 3. Analysis of the aggregation behavior of NanA and NanA-C. SEC
analysis showed the formation of protein aggregates only at high
concentrations of 125 µM (blue curve) and a unique peack (Vr = 84 mL) at
13µM (grey curve) and 64 µM (orange curve).
Next, we used SPR to study the faculty of the compounds 1-9
to disrupt NanA adherence in a competition assay. Inhibition
constants expressed per mol of sialosides (KIsi) are reported in
Figure 4B. This SPR assay reflects the relative affinity of the
compounds for the NanA constructs which differs from and
complements the measurement of the inhibition of the catalytic
enzyme activity (Table 1). Divalent compounds 2-8 showed a
moderate affinity improvement with a maximal RIP value of
around 5 (compound 4 KIsi = 9.4 µM for NanA). Again, the most
potent compound was polymer 9 showing KIsi values of 3.1, 61
and 2.3 µM for NanA, NanA-C and NanA-L, respectively.
Although significant, this improvement in terms of affinity
remains moderate compared to the very high improvement in
inhibiting the catalytic activity of NanA (RIP = 2927) and NanA-
C (RIP = 2979). The two assays measure very different
characteristics of the enzymes, which are the inhibition of its
hydrolyzing activity and its binding potency. In the SPR assay,
the compounds should prevent and disrupt NanA attachment
from a heavily functionalized thiosialoside surface.
All the compounds, including 9 showed much weaker affinity
for NanA-C compared to NanA-L or NanA. Coherently to the
Kd values obtained with the sialylated SPR surface alone, the
lectin domain NanA-L was shown to improve the polymer
binding for Nan-A significantly. The highly similar KIsi recorded
for the compounds with NanA and NanA-L but not NanA-C
(Figure 4B) suggests that they predominantly bind to the lectin
domain.
Thus a significant quantity of NanA-C aggregates is only
partially formed at the high protein concentration of 125 µM (7
mg/ml). Consequently, self-associative processes are unlikely
during the measurement of the inhibitory activity due to the
very low protein concentration used (~ 1-5 nM).
Binding affinity by Surface plasmon resonance (SPR). We
studied the affinity of NanA, NanA-C and NanA-L for a surface
coated with thiosialosides mimicking cell-surface sialoglycans,
using surface plasmon resonance (SPR). Thiosialoside 34
(structure and synthesis in SI) appended with a terminal amino-
group instead of a primary alcohol was coated through the
formation of an amide bond. Dissociation constants from the
three constructs highlighted the benefit of the lectin domain to
improve NanA affinity for a sialylated surface (Figure 4A).
Indeed, NanA showed a 283-fold lower dissociation constant
(Kd = 0.755 µM) compared to NanA-C (Kd = 214 µM). This
affinity improvement is mainly due to the higher intrinsic affinity
of NanA-L (Kd = 10.3 µM) for the sialylated surface. Thus,
NanA binding to a sialylated surface is mainly promoted by the
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Figure 4. A) Illustration of the SPR assay. 1) The dissociation constants of
the NanA constructs for the thiosialylated surface were first measured
without compounds 2) Competitive experiments were subsequently
performed to determine Kisi values for compounds 1-9. B) Inhibition values
(Kisi) expressed in µM of thiosialoside residues.
Figure 5. A) Illustration of the chip assay. NanA constructs were grafted on
the plate and the interaction with thiosialylated BSA was measured.
si
Competitive experiments were subsequently performed to determine IC50
values for compounds 1-9 (reported in Table 1). B) Binding inhibition of
NanA, NanA-C and NanA-L for the thiosialylated BSA.
Binding affinity on chip. The binding inhibition of the
compounds 1-9 was subsequently evaluated using a very
different system adapted from “lectin glycoprofiling”
technologies.[37] First, NanA constructs were coated on a
microplate surface. Labelled-BSA coated with azido-
functionalized thiosialosides by click-chemistry (see supporting
information for detailed procedure) was then added to the plate
(Figure 5A). In the first set of experiments serial dilutions of
thiosialylated BSA were applied and the recorded absorbance
determined the interaction profiles with NanA, NanA-C and
NanA-L. As seen in Figure 5B NanA and NanA-L have very
similar inhibition profiles and exceed by around two orders of
magnitude NanA-C affinity for the thiosialylated-BSA. This is
fully coherent with the SPR results and further highlights the
key role of lectin domain in driving the sialoside binding.
Competitive experiments with serial dilutions of compounds 1-
9 were performed to obtain IC50 values (Table 1). The binding
assay could not be performed with NanA-C due to its much
lower affinity for the thiosialylated-BSA probe. Monovalent
reference 1 was shown to be a significantly more potent
inhibitor for NanA and NanA-L compared to Neu5Ac,
highlighting the positive impact of the anomeric residues of 1.
Significant synergistic effects were not observed with divalent
Si
2-8. All compounds showed poor IC50 values ranging from
440 to 2160 µM. In contrast polymer 9 showed low IC50si values
of 3.5 and 2.3 µM for NanA and NanA-L, which represents high
RIP values of 254 and 939-fold, respectively. Again, the
polymer binding affinity for the sialidase is largely promoted by
si
the lectin domain in view of the nearly identical IC50 values
observed for NanA and NanA-L.
High levels of affinity improvements have been previously
reported with glycopolymers and lectins.[20] We anticipated that
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polymer 9 could also act as a potent inhibitor of sialoside-
binding lectins. We selected Wheat Germ Agglutinin (WGA) as
a model lectin, which was previously shown to be successfully
inhibited by multivalent N-acetylglucosamine derivatives.[14,38]
As seen in Table 1, WGA can also accommodate sialosides
such as Neu5Ac and 1, and with a similar range of affinity
(mM) compared to NanA and NanA-L. In the divalent series 2-
0.755 µM, NanA-C kd = 214 µM). A similar scenario was
observed when the NanA constructs were immobilized on a
chip. The affinity profile for the thiosialylated BSA was nearly
identical for NanA and NanA-L, and two-orders of magnitudes
lower for NanA-C (Figure 5B). Thus, the binding to a sialylated
surface is almost exclusively promoted by the lectin domain.
The blend of assays also shed light on the binding interaction
with the polymer 9. Results obtained from the binding inhibition
experiments with 9 showed high and very similar binding
potency against NanA and NanA-L with KIsi and IC50si values of
3.1 vs 2.3 and 3.5 vs 2.3 µM, respectively. The binding of the
polymer to NanA is also fully promoted by the lectin domain
and a chelate binding mode with thiosialosides ligands
embracing both the catalytic and lectinic domains, as initially
hypothesized, seems irrelevant. Unexpectedly, 9 exerted very
high inhibition of the enzymatic activity (Figure 2B, 2C) on both
NanA and NanA-C (KIsi = 409 ± 65 nM and 335 ± 40 nM). This
suggests that the activity of sialidases lacking a lectin domain
can be efficiently inhibited by polymeric thiosialosides such as
9. The strong inhibition observed did not result from NanA-C
aggregation as evidenced by DLS, SEC and MALS. We
propose that the enhanced affinity and inhibitory activity
observed with 9 results from a “bind and jump” process were
the NanA constructs diffuse from one thiosialoside ligand to
another along the polymeric chain, as previously described by
Brewer on GalNAc binding lectins.[23] This “zipping”
mechanism is not accessible to divalent compounds 2-8 that
are indeed poor sialidase inhibitors.
si
8, the IC50 values were shown to gradually decrease from
4300 µM to 130 µM when the spacer arm length was increased
from 2 to 130 EG units. This significant affinity improvement
was, however exceeded by polymer 9 showing IC50si of 1.2 µM
and calculated RIP values 917, respectively. Thus, polymer 9
showed similar and high levels of affinity improvement on WGA,
NanA and NanA-L, blurring the line between the lectins and
sialidases.
Table 1. Binding inhibition against NanA and NanA-C determined on a chip.
Cpds
V(n)
1
WGA
9500 ± 500 8420 ± 100
1100 ± 100 890 ± 7
4300 ± 200 800 ± 200
NanA
NanA‐L
Neu5Ac
6840 ± 300
2160 ± 200
1560 ± 80
1200 ± 140
540 ± 200
480 ± 180
520 ± 40
1
1(2)
2
3
4
5
6
7
8
9
2(2)
2(10)
2(30)
2(43)
2(66)
2(86)
2(130)
1500 ± 200
540 ± 40
330 ± 40
320 ± 20
182 ± 10
880 ± 200
440 ± 160
760 ± 40
720 ± 100
580 ± 80
1040 ±140
3.5 ± 0.3
To summarize, the design of polymeric thiosialosides led to
highly potent sialidase inhibitors with high sygnergistic
multivalent effects. Binding affinities were significantly
increased with sialidase lectin domains, but strong inhibition of
the enzymatic activity was observed on the catalytic domain.
560 ± 200
780 ± 200
2.3 ± 0.2
130 ± 10
The design of multivalent glycosidase modulators is
a
promising emerging field[39–43] as illustrated by the continuous
reports of potent glycosidase activators[44,45] or inhibitors based
on clusterized iminosugars [46,46–49] or sugar substrates.[50] This
concept is now extended to the inhibition of bacterial and
pathogenic sialidases for which transition-state inhibitors fail to
reach the submicromolar level.
130(2) 1.2 ± 0.3
Conclusions
We have designed non-hydrolysable multivalent thio-
sialosides as probes and inhibitors of biologically relevant
sialidases. The polymeric compound 9 was by far the most
active showing unprecedented levels of inhibitory capacity and
binding affinity against the biologically relevant TcTS and
NanA sialidases. Each thiosialoside ligand attached to the
polymer backbone surpassed the inhibitory capacity of the
corresponding monovalent reference by more than three
orders of magnitude. Thus, polymeric thiosialosides and
sialidases can reach the high level of inhibition previously
reported for glycopolymers and lectins. This is relevant
considering the difficulty in designing potent sialidases and
trans-sialidases inhibitors. Importantly, the expression of the
NanA truncated domains NanA-C and NanA-L allowed a better
understanding of the respective functions of the catalytic and
lectinic domains of the pathogenic sialidases. The lectinic
domain improves NanA binding to a sialylated SPR surface by
Acknowledgements
This work was carried out with financial support from the the
Centre National de la Recherche Scientifique (CNRS), the
Ministère de l’Enseignement Supérieur et de la Recherche in
France, the National Agency for Research (ANR project
HICARE 17-CE07-028-01) and the Conseil Régional des Pays
de la Loire (MAGGIC project). BD is funded by the European
Union’s Horizon 2020 research and innovation program (Marie
Sklodowska-Curie grant agreement No. 642870, ETN-
Immunoshape).
Keywords: Sialidases • Multivalency • Glycoclusters•
Glycosidases • Enzymatic inhibition
more than two orders of magnitude (Figure 4A, NanA kd
=
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Entry for the Table of Contents
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High synergy: Sialidases (SA) are
virulence factors expressed by
pathogenic bacteria, viruses and
parasites. Di- and polymeric
thiosialosides were designed to inhibit
V. cholerae, T. cruzi and S.
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
pneumoniae sialidases. Each
thiosialoside grafted on the polymer
was shown to reduce SA catalytic
activity with much higher efficiency
compared to their monovalent
analogues. This extend the multivalent
concept to this class of enzymes.
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