Dynamic Cooperative Glycan Assembly Blocks the Binding of Bacterial Lectins to Epithelial Cells

Article abstract of DOI:10.1002/anie.201700813

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell-surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA–fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand–protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700-fold compared to l-fucose.

Full text of DOI:10.1002/anie.201700813

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201700813
German Edition: DOI: 10.1002/ange.201700813
Glycoconjugates
Hot Paper
Dynamic Cooperative Glycan Assembly Blocks the Binding of
Bacterial Lectins to Epithelial Cells
Takuya Machida, Alexandre Novoa, ꢀmilie Gillon, Shuangshuang Zheng, Julie Claudinon,
Thorsten Eierhoff, Anne Imberty,* Winfried Rçmer,* and Nicolas Winssinger*
Abstract: Pathogens frequently rely on lectins for adhesion
and cellular entry into the host. Since these interactions
typically result from multimeric binding of lectins to cell-
surface glycans, novel therapeutic strategies are being devel-
oped with the use of glycomimetics as competitors of such
interactions. Herein we study the benefit of nucleic acid based
oligomeric assemblies with PNA–fucose conjugates. We dem-
onstrate that the interactions of a lectin with epithelial cells can
be inhibited with conjugates that do not form stable assemblies
in solution but benefit from cooperativity between ligand–
protein interactions and PNA hybridization to achieve high
affinity. A dynamic dimeric assembly fully blocked the binding
of the fucose-binding lectin BambL of Burkholderia ambifa-
ria, a pathogenic bacterium, to epithelial cells with an efficiency
of more than 700-fold compared to l-fucose.
ization of dynamic fucose assembly operates cooperatively
with ligand binding.
The Burkholderia cepacia complex is a group of closely
related bacteria that cause lung infections in immunocom-
promised patients as well as in patients with granulomatous
disease or cystic fibrosis.[5] Burkholderia ambifaria lectin
(BambL) and Ralstonia solanacearum lectin (RSL) are
bacterial lectins that share a six-bladed beta-propeller fold
formed by trimerization of monomers carrying two fucose-
binding sites, resulting in six fucose binding sites on the same
face (Figure 1).[5,6] It has been shown that the interaction of
RSL with fucosylated epitopes such as histo-blood group
T
he role of multivalent interactions at cellular interfaces is
well recognized.[1] Frequently, these interactions involve cell-
surface glycans, which are specifically recognized by protein
receptors such as lectins. Such interactions are generally of
low affinity, but a high overall avidity is achieved through
mutivalency. Pathogens frequently harness the specific rec-
ognition of host epithelial glycans for adhesion and infection
with multivalent lectins,[2–3] and inhibition of these interac-
tions by competition with soluble glycocompounds represents
a promising therapeutic avenue.[4] Herein, we investigated the
gain of affinity achieved with nucleic acid based assemblies to
oligomerize l-fucose, and we demonstrate that the hybrid-
[*] T. Machida, Dr. A. Novoa, Prof. N. Winssinger
Department of Organic Chemistry, NCCR Chemical Biology
Faculty of Science, University of Geneva
Quai Ernest Ansermet 30, 1211 Geneva (Switzerland)
E-mail: nicolas.winssinger@unige.ch
ꢁ. Gillon, Dr. A. Imberty
CERMAV UPR5301, CNRS, and Universitꢂ Grenoble Alpes, BP 53
38041 Grenoble cedex 9 (France)
E-mail: anne.imberty@cermav.cnrs.fr
S. Zheng, Dr. J. Claudinon, Dr. T. Eierhoff, Prof. W. Rçmer
Faculty of Biology, and Centre for Biological Signalling Studies
(BIOSS), Albert-Ludwigs-University Freiburg
Schꢃnzlestraße 18, 79104 Freiburg (Germany)
E-mail: winfried.roemer@bioss.uni-freiburg.de
Dr. T. Eierhoff
Present address: Institute of Biochemistry
Heinrich-Heine-University Dꢄsseldorf
Universitꢃtsstraße 1, 40225 Dꢄsseldorf (Germany)
Figure 1. Space-filling and ribbon representations of the crystal struc-
ture of RSL in complex with a-methyl-l-fucoside represented as a wire
frame (PDB ID: 2BT9). Blue arrows indicate the 20 ꢀ through-space
distance between the binding sites. RSL=Ralstonia solanacearum
lectin.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201700813.
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oligosaccharides exposed on
glycolipids is sufficient to
induce membrane invagina-
tions in giant liposomes.[7,8]
Furthermore,
oligomeric
interactions are important
for this invagination, since
mutated lectins bearing only
three fucose binding sites
(e.g., the R17A mutant)
also interacted with lipo-
somes but failed to yield
invagination. Based on the
interest in using nucleic acid
hybridization of glycan-
PNA conjugates (PNA =
peptide nucleic acid)[9–16] to
tailor assemblies that can
bridge multiple binding
sites in target proteins, we
sought to evaluate the ben-
efit of oligomeric assemblies
for RSL and ultimately, to
outcompete the interaction
of such lectins with epithe-
lial cells.
We began our investiga-
tion with RSL, designing
a fucose ligand flanked by
two 8-mer PNA (Figure 2).
Using the appropriate com-
plementary sequences, the
fucose ligand can be oligo-
merized incrementally to
a
cyclic hexamer that
would form a ring around
the protein. The 8-mer PNA
duplex stretches over 34 ꢀ
Figure 2. A) Structure of the PNA–glycan adduct (top, PNA color denotes self-complementarity, red triangle
denotes fucose) and SPR-measured KD for the assemblies. B) SPR sensorgrams for Fuc-triazole injected onto
RSL chips with concentrations starting at 3.33 mm and diluted by 3-fold at each step. C) The same
experiments with the assembly produced from the mixture of compound 1 and 2, which form a dimer
(starting concentration 1 mm). D) The same experiment with compounds 1 to 6, which form a hexavalent
assembly (starting concentration 0.1 mm). In all cases the bottom line corresponds to control injection of
buffer. The binding kinetics of the experiments were too fast (Fuc-triazol) or too complex (multivalent) to
evaluate dissociation constants, and steady-state analysis was performed instead. PNA=Peptide nucleic acid;
RSL=Ralstonia solanacearum lectin; SPR=surface plasmon resonance.
with
(through-space
a
small curvature
distance:
PDB ID: 3PA0)[17] to yield
a hexagone with a perimeter
of 204 ꢀ, which was deemed
sufficiently large to fit the
protein. The fucose ligand is
linked to each side of the
PNA with a 10 ꢀ flexible
PEG spacer that leave some plasticity to fit the multivalent
interaction. The 8-mer PNA have Tm > 608C, thus yielding
stable assemblies at room temperature. The affinities of the
different assemblies were measured by surface plasmon
resonance (SPR) using a chip with low protein loading to
avoid artifacts from assemblies bridging two proteins. The
control l-fucose–triazole conjugate (Fuc-triazole, Figure 2)
has an affinity for RSL of approx. 2 mm, which is in the same
range as that previously reported for fucose.[6] The presence of
PNA on both sides of fucose increases the affinity for the
lectin by one order of magnitude compared to the Fuc-
triazole control compound, resulting in a KD of 362 nm,
however, the PNA duplex lacking fucose did not show
measurable affinity to the lectin. As shown in Figure 2,
a significant gain in affinity is obtained in the progression
from monomeric ligand with flanking double strand PNAs
(362 nm) to the dimeric one (3.3 nm). This gain in affinity is
consistent with the chelate effect of divalent interactions and
confirmed that the 8-mer PNA with the flexible PEG linker is
able to bridge two binding sites. However, progression to
higher-order oligomers only brought small incremental bene-
fit (3 nm for the trimer, 1 nm for the tetramer, 0.9 nm for the
pentamer and 0.5 nm for the cyclic hexamer). Comparison of
SPR sensorgrams obtained with monomeric and hexameric
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compounds is displayed in Figure 2 (See Figure S1 in the
mer PNA using two different sequences with different duplex
Supporting Information for complete set of sensorgrams),
thus illustrating the strong gain in affinity observed for
oligomers. Enthalpy–entropy compensations have frequently
been attributed to diminishing return in affinity gain with
higher-order oligomeric interactions. Nonetheless, the sub-
nanomolar affinity for the hexameric assembly remains
notable considering that fucose ligand by itself has an affinity
of 2200 nm (i.e., there is a > 1000 fold gain in affinity).
We next asked whether the significant gain in affinity
observed for the static dimeric assembly could be achieved
with shorter PNA that would not form stable duplexes in
solution but only in the presence of the lectin through the
cooperative interaction of the PNA duplex and the ligand–
target interactions (Figure 3). To this end, we prepared the
same fucose ligand with self-complementary palindromic 4-
stabilities (7a (TTAA): KD = 13 mm, Tm < 158C at 2 mm; 7b
(GGCC): KD = 3.8 mm, Tm = 238C at 2 mm; Figure S2,3). Thus
at concentrations below 1 mm, the ligands 7a and 7b are
predominantly in the dissociated monomeric form. However,
the duplex should be stabilized by the high effective concen-
tration of the PNA at the surface of the lectin and the lectin
binding should be enhanced through the chelate effect of the
hybridized PNA. As shown in Figure 3, while the fucose
ligand has an affinity of 2.2 mm, ligand 7a has an affinity of
83 nm and ligand 7b has an affinity of 22 nm. When comparing
the dissociation phase in the SPR sensorgrams, the fucose–
triazole adduct lacking the PNA has a very fast off-rate (koff
=
2 ꢁ 10À1 sÀ1, Figure 2B), while the assembly arising from 7b
dissociates much more slowly (koff = 5 ꢁ 10À4 sÀ1, Figure 3
bottom). We therefore observe a clear increase in the
Figure 3. Top: Schematic representation of the dynamic fucose dimer interacting with RSL. Green=RSL, red triangle=fucose; Structures of
compounds 7–9 and affinities for assemblies prepared from 7a and 7b. Bottom: SPR sensorgrams for compound 7b injected on RSL chips with
concentrations starting at 6 mm and diluted by 3-fold at each step; derivation of the dissociation constant from steady-state analysis.
PNA=peptide nucleic acid; RSL=Ralstonia solanacearum lectin; SPR=surface plasmon resonance.
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residence time at concentrations well below duplex formation
with a very slow dissociation phase that is characteristic of
multivalent interactions. As further controls, we prepared the
equivalent of compound 7b with a glucose instead of fucose
(8) or omitting conjugation with a carbohydrate (9). Neither
of these controls had measurable affinities for the lectin
(RSL). To further assess the cooperative dynamic assembly of
7b, we performed SPR measurements of 7b binding to RSL in
the presence of increasing amounts of control 9 which can
compete for hybridization (up to 5 equivalents). The presence
of control 9 had no impact on the affinity of 7b at
stoichiometric amounts (at this concentration, the probability
of a 7b dimer from random hybridization is 25%; see
Figure S1 for the sensorgram). At higher concentrations (2.5
and 5 equivalents), the binding curves were slightly altered
due to non-specific binding of 9 in the control channel,
however, the same slow dissociation was observed as in the
binding of 7b alone (at 5 equivalent of 9, the statistical
probability of a 7b dimer is 2.7%), thus suggesting a self-
sorting of 7b dimer on the protein. The dramatic gain in
affinity observed for the dynamic assemblies of 7a or 7b, as
well as the lack of influence of 9 in competing for hybrid-
ization, can only be rationalized by the cooperativity of PNA
hybridization and lectin interaction wherein the rebinding of
ligand in the dimeric chelate is faster than PNA–duplex
dissociation, and likewise, duplex formation at the protein
surface is faster than koff of the fucose ligand.
with the Fuc-triazole lacking the PNA did not show any
activity at concentrations up to 100 mm and did not reach
complete inhibition at the highest tested concentration (1 mm,
Figure S4). The gain of efficacy observed in this assay with the
dynamic assemblies concurs with affinity measurements on
RSL. It should be noted that the assays were carried out at
a RSL concentration of 178 nm with a capacity to bind up to
1 mm of fucose ligand. While occupancy of the six binding sites
is not necessary for adhesion inhibition, the IC50 measurement
points to the fact that the inhibition is achieved with nearly
stoichiometric quantities of ligands relative to the lectin.
Furthermore, these cellular assays were performed at 378C,
which further displaces the equilibrium away from duplex
formation. The fact that assemblies 7b were more efficacious
than the ligand alone at this temperature indicates that the
cooperativity between ligand binding and duplex formation is
still operative.
Multivalency plays a major role in biological processes
and is omnipresent in the interaction of pathogenic micro-
organisms with their host. The search for high-affinity ligands
that can inhibit these interactions has predominantly focused
on oligomeric, polymeric, and dendritic scaffolds functional-
ized with multiple ligand copies.[4,18] To the best of our
knowledge, this is the first example that demonstrates that
such interactions can be inhibited with dynamic supramolec-
ular assemblies in a cellular context. While dynamic combi-
natorial chemistry has been investigated for drug discovery
and receptors using reversible covalent chemistry,[19,20] the use
of hybridization can be tuned to yield the benefit of
cooperativity between the ligand pairing through base pairing
and the chelate effect of the ligand interactions with the target
that is not achievable with covalent dynamic assemblies. This
concept has inspired elegant studies to pair DNA- or PNA-
encoded organic fragments for drug discovery.[21–26] In the
present case, the dynamic assembly was 723-fold more
effective than the l-fucose alone in a cell-based assay. Many
other pathogenic bacteria (e.g., P. aeruginosa via LecA or
LecB, or uropathogenic E. coli via FimH) and bacterial
products (e.g., Shiga toxin) that interact with host-cell
glycoreceptors could in principle be cleared by dynamic
cooperative glycan assembly.
We then investigated the efficacy of the more potent
assembly 7b to block the binding of BambL to H1299 lung
epithelial cells. For this purpose, we prepared both anomeric
configurations of the fucose linked to the PNA. As shown in
Figure 4, assemblies arising from both anomeric configura-
tions were competent in inhibiting BambL binding to
epithelial cells in a dose-dependent manner with IC50 values
of 0.56 mm and 0.94 mm for the b-anomer and a-anomer of
fucose, respectively. Impressively, assembly 7b (b-anomer)
was 723-fold more effective than the fucose alone. Assays
Acknowledgements
This work was supported by CNRS, Universitꢂ Grenoble
Alpes, Labex ARCANE (ANR-11-LABX-0003-01), Swiss
National Science Foundation (SNSF) and the NCCR Chem-
ical Biology for financial support. A.I. and W.R. acknowledge
support by the French National Research Agency (ANR) and
the German Federal Ministry of Education and Research
(BMBF) in the framework of the EU ERASynBio project
SynGlycTis (ANR-14-SYNB-0002-02; BMBF 031A464).
W.R. was supported by the Excellence Initiative of the
German Research Foundation (EXC 294), the Ministry of
Science, Research and the Arts Baden-Wꢃrttemberg (Az: 33–
7532.20), and by a starting grant from the European Research
Council (Programme “Ideas”, ERC-2011-StG 282105). S.Z.
acknowledges support from the China Scholarship Council.
Figure 4. Inhibition of BambL binding to H1299 lung epithelial cells.
Left: Dynamic assembly arising from 7b with the a-l-fucose conjugate.
Middle: Dynamic assembly arising from 7b with the b-l-fucose
conjugate. Right: Data for l-Fucose (corresponding bar charts with
error bars are provided in Figure S4). BambL=Burkholderia ambifaria
lectin.
4
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Manuscript received: January 23, 2017
Revised: March 20, 2017
Final Article published: && &&, &&&&
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Communications
Glycoconjugates
Short and sweet: The interactions of
lectins (blue) with cells can be inhibited
with nucleic acid based oligomeric
T. Machida, A. Novoa, ꢁ. Gillon, S. Zheng,
J. Claudinon, T. Eierhoff, A. Imberty,*
W. Rçmer,*
assemblies with PNA–fucose conjugates.
These conjugates do not form stable
assemblies in solution but benefit from
cooperativity between ligand–protein
interactions and PNA hybridization. A
dynamic dimeric assembly fully blocked
the binding of the fucose-binding lectin
BambL of the pathogenic bacterium
Burkholderia ambifaria to epithelial cells.
N. Winssinger*
&&&& — &&&&
Dynamic Cooperative Glycan Assembly
Blocks the Binding of Bacterial Lectins to
Epithelial Cells
6
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