Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging

Article abstract of DOI:10.1002/anie.201709368

Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing “pathoblockers” preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.

Full text of DOI:10.1002/anie.201709368

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A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Covalent lectin inhibition and application in bacterial biofilm
imaging
Authors: Stefanie Wagner, Dirk Hauck, Michael Hoffmann, Roman
Sommer, Ines Joachim, Rolf Müller, Anne Imberty, Annabelle
Varrot, and Alexander Titz
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10.1002/anie.201709368
Angewandte Chemie International Edition
Covalent lectin inhibition and application in bacterial biofilm imaging
Stefanie Wagner1,2, Dirk Hauck1,2, Michael Hoffmann2,3, Roman Sommer1,2, Ines Joachim1,2, Rolf
Müller2,3, Anne Imberty4, Annabelle Varrot4, Alexander Titz1,2*
1 Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), D-66123 Saarbrücken, Germany
2 Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover–Braunschweig (Germany)
3 Microbial Natural Substances, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
D-66123 Saarbrücken, Germany
4 Université Grenoble Alpes, CERMAV, F-38000 Grenoble, France; CNRS, CERMAV, F-38000
Grenoble, France.
corresponding author, email: alexander.titz@helmholtz-hzi.de
*
1
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10.1002/anie.201709368
Angewandte Chemie International Edition
Abstract
Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins
play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting
various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm
building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in
developing ‘pathoblockers’ preventing biofilm formation and virulence. Here, we describe for the
first time a covalent lectin inhibitor specific to a carbohydrate binding site. In addition we report its
application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa.
Lectins are carbohydrate-binding proteins with very diverse functions that are found in all domains
of life.[1] These proteins play crucial roles in numerous processes such as cell-cell recognition,
infection processes and immune defense. They are generally characterized by an intermediate to
low affinity towards their carbohydrate ligands that is often overcome by Nature through
multivalency of both the lectin receptors and their carbohydrate ligands resulting in avidity with an
increase in apparent affinity.
Because these carbohydrate-binding proteins play essential roles in a number of pathological
processes, they have become attractive targets for therapy. However, the fact that lectins display
moderate affinities to their ligands renders this class of proteins as difficult targets for drugs.[2]
Despite this drawback, a number of recent success stories impressively demonstrated their potential
for therapy: the selectin antagonist GMI-1070 is currently in phase III clinical trials and various
FimH inhibitors are in the late preclinical stage.[2, 3]
Lectins are involved in infections with the Gram-negative bacterium P. aeruginosa, one important
member of the often highly drug resistant ESKAPE pathogens which are Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, P. aeruginosa, and
Enterobacter species and currently cause most of the severe hospital infections in western countries.
[4]
The two bacterial lectins,[5] LecA and LecB, are virulence factors and important for bacterial
adhesion and biofilm formation.[6] The latter is especially problematic as resistance against
antibiotics inside a biofilm is increased by a factor of 10-1000.[7] Thus, the inhibition of these
lectins provides a promising way to dismantle the bacterium from the protective biofilm
environment and restore immune defense and activity of antibiotics.[8] Current approaches to inhibit
both lectins range from small molecules to multivalent structures and are summarized in recent
2
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10.1002/anie.201709368
Angewandte Chemie International Edition
reviews.[9] We focus on the development of small molecules and recently published various potent
glycomimetic inhibitors for the high affinity lectin LecB as inhibitors of P. aeruginosa adhesion.[10]
In contrast, LecA only has an intermediate affinity for its monovalent D-galactose-derived ligands in
the 50-100 µM range.[5,11] Phenyl β-D-galactosides and derivatives showed an increased affinity of
approx. 10 µM, e.g. compound 1 (Figure 1), but despite a high number of derivatives analyzed, no
further significant increase in potency could be achieved.[12]
Covalent inhibition is one strategy to avoid dissociation of the inhibitor from the target and thus to
persistently inactivate proteins. To date, a specific covalent inhibition of the carbohydrate binding
site in a lectin has not been achieved despite attempts using squaric acid to target FimH[13] or
photoactivatable substituents for the targeting of galectins.[14] The latter probes covalently bind to
unspecific residues of the protein in proximity (3 Å) to the photoactivated center. The crystal
structure of LecA[11a,15] reveals the presence of one cysteine residue (Cys62) in the carbohydrate
binding domain (Figure 1). The specific targeting of cysteine residues with electrophilic warheads is
a general strategy in the search for cysteine protease inhibitors,[16] but has never been addressed in
carbohydrate recognition domains. In order to target Cys62, we designed the two diastereoisomeric
galactose-derived epoxides 2 and 3 (Figure 1) as potential covalent active site inhibitors of LecA.
A.
Asn107
NH2
NO2
1
O
Thr104 (bb)
Asn108 (sc)
HO
O
HO
HO
O
Ca2+
His50(sc)
OH
Cys62 SH
O
Asp100
O
H2O
Gln53(sc/bb)
Tyr36
(sc)
(bb)Pro51
B.
HO
HO
O
HO
HO
O
HO
HO
O
O
O
O
2
3
Figure 1: A. Galactoside recognition by the bacterial lectin LecA (pdb: 3ZYF[15]); B. Electrophilic epoxide derivatives
2 and 3 for the targeting of Cys62. The distances from Cys62-S to C6 and O6 of 1 are between 4.1 and 4.3 Å. (sc = side
chain, bb = back bone)
3
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