Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas aeruginosa

Article abstract of DOI:10.1021/jacs.7b11133

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.

Full text of DOI:10.1021/jacs.7b11133

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Article
Glycomimetic, orally bioavailable LecB inhibitors
block biofilm formation of Pseudomonas aeruginosa
Roman Sommer, Stefanie Wagner, Katharina Rox, Annabelle Varrot, Dirk Hauck, Eike-
Christian Wamhoff, Janine Schreiber, Thomas Ryckmans, Thomas Brunner, Christoph
Rademacher, Rolf W. Hartmann, Mark Brönstrup, Anne Imberty, and Alexander Titz
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11133 • Publication Date (Web): 22 Dec 2017
Downloaded from http://pubs.acs.org on December 22, 2017
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Glycomimetic, orally bioavailable LecB inhibitors block biofilm formation of
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Pseudomonas aeruginosa
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Roman Sommer1,2, Stefanie Wagner1,2, Katharina Rox2,3, Annabelle Varrot4, Dirk Hauck1,2, Eike-
Christian Wamhoff5,6, Janine Schreiber2,7, Thomas Ryckmans8, Thomas Brunner9, Christoph
Rademacher5,6, Rolf W. Hartmann2,10,11, Mark Brönstrup2,3, Anne Imberty4, and Alexander Titz1,2,11*
1Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), Helmholtz Centre for Infection Research (HZI), D-66123 Saarbrücken, Germany
2Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig, Germany
3Chemical Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany
4Centre de Recherche sur les Macromolécules Végétales (CERMAV-UPR5301) CNRS and
Université Grenoble Alpes, BP53, 38041 Grenoble cedex 9, France
5Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, D-14424
Potsdam, Germany
6Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, D-14195 Berlin,
Germany
7Infection Immunology, Helmholtz Centre for Infection Research, D-38124 Braunschweig,
Germany
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8Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, CH-
4070 Basel, Switzerland
9Biochemical Pharmacology, Department of Biology, University of Konstanz, D-78457 Konstanz,
Germany
10Drug Design and Development, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Helmholtz Centre for Infection Research (HZI), D-66123 Saarbrücken, Germany
11Department of Pharmacy, Saarland University, D-66123 Saarbrücken, Germany
* Corresponding author
Tel. +49 681 98806 2500
email: alexander.titz@helmholtz-hzi.de
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Abstract
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The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for
infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to
switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism
of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral
component and necessary for biofilm formation. Here, we report a new class of drug-like low
molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor
binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked
biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands
failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably,
two compounds showed good oral bioavailability and high compound concentrations in plasma and
urine were achieved in vivo.
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Introduction
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The Gram-negative bacterium Pseudomonas aeruginosa (P. aeruginosa) has become a severe threat
for hospitalized immuno-compromised patients and is now a critical priority 1 pathogen as stated by
the WHO in 2017.1-4 In persons suffering from cystic fibrosis, chronic infections with this pathogen
lead to recurrent pneumonia, sepsis and lung damage.5 Infections of various organs and tissues by
this pathogen, such as chronic wound6 or catheter-associated urinary tract infections7 (caUTI),
impose severe clinical challenges which further result from the bacterium’s intrinsic drug resistance
and additional acquired resistances often leading to multi or extreme drug resistant strains (MDR,
XDR).8 Furthermore, the antimicrobial tolerance of P. aeruginosa is enhanced through formation of
biofilms, a self-made environment that protects bacteria against host immune defense and antibiotic
treatment.9,10 Triggered by the fact that bacteria inside a biofilm are up to 1000-fold more resistant
towards antibiotics,9 the targeting of biofilm formation as therapeutic approach emerged in order to
overcome the resistance problem (reviewed in11,12).
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The two quorum sensing regulated13 virulence factors LecA14 and LecB15 (formerly called PA-IL
and PA-IIL) are crucial for biofilm formation by P. aeruginosa. These two carbohydrate-binding
proteins, i.e. lectins, were initially isolated from the clinical isolate PAO1 by Gilboa-Garber et al..16
Problematic for therapy, P. aeruginosa shows a high genomic diversity among different isolates.17,18
While LecA is relatively conserved among strains, LecB varies and clinical isolates can be grouped
into either PAO1- or PA14-like LecB sequence families, and both types cover approximately 50%
each of the clinical isolates.19,20 However, we could show that despite numerous sequence variations
in the two LecB variants, they possess comparable binding specificity which is a prerequisite for
targeting a broad range of clinical isolates with one single drug.20
The exact mechanism of the lectins’ role in biofilm formation is not fully understood. It is assumed,
that these tetravalent lectins act as bridging agents to enable glycan-mediated bacterial adhesion to
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the cellular glycocalix of the host’s tissue. In addition, they are believed to mediate contact between
bacterial cells through cell-surface glycoconjugates and, further, with the bacterially produced
multivalent exopolysaccharides for the establishment of bacterial aggregates and biofilms.11 This
biofilm reinforcing property of the lectins, which results from its multivalency, can be blocked by
inhibition of the carbohydrate binding sites.
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In a mouse infection model, LecB was shown to be the major determinant for bacterial lung
colonization.21,22 Furthermore, this protein influences signaling pathways in human lung epithelial
cells leading to β-catenin degradation, a host protein that is crucially involved in tissue repair.23 On
a structural level, LecB forms non-covalent homotetramers and each monomer contains two Ca2+-
ions,20,24 which mediate the binding to its carbohydrate ligands, L-fucose and D-mannose. It has been
shown in humans and mice, that inhalation of millimolar concentrations of these natural sugars
contributed to clearance of bacteria from the lungs and, furthermore, showed synergistic activities
with co-administered antibiotics.25,26 Due to higher affinities towards fucosides over mannosides,
research focussed on fucose-based inhibitors with multivalent presentation to further increase
avidity.27,28 This approach yielded multivalent glycopeptide dendrimer structures with the ability to
efficiently inhibit the formation of and disperse established biofilms of P. aeruginosa.29
Interestingly, in another case of multivalent fucosides, despite nanomolar affinities for LecB,
millimolar concentrations (5 mM) have been reported necessary for efficient inhibition of biofilm
formation and compounds were inactive at 100 µM.21 In the same study, these multivalent inhibitors
were even shown to promote bacterial aggregation. Thus, it is conceivable that some multivalent
lectin inhibitors can act as mimics of the bacterial exopolysaccharides and, therefore,
unintentionally contribute to biofilm integrity rather than to its desired disintegration.
In order to circumvent such a detrimental biofilm-stabilizing effect of multivalent ligands through
cross-linking of the tetravalent lectins inside the biofilm, we have focussed on monovalent low
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molecular weight inhibitors of LecB.30-34 In addition, only small molecules can be developed into
drug-like molecules with favorable pharmakokinetic properties, i.e. oral application with systemic
drug bioavailability, which is necessary to reach all possible types of the various organ and tissue
infections elicited by P. aeruginosa. Thus, small monovalent molecules hold the premise to
circumvent both biofilm-reinforcing effects and the restriction to topical applications for the
multivalent systems.
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Here, we report two types of monovalent glycomimetic LecB inhibitors, cinnamides 6 and
sulfonamides 7. These new low molecular weight C-glycosidic LecB inhibitors showed nanomolar
potency against LecB with excellent target selectivity, toxicity and in vitro ADME properties.
Notably, the glycomimetic sulfonamides developed here were potent inhibitors of biofilm formation
in stark contrast to the ineffective native carbohydrates. Finally, a murine in vivo pharmacokinetic
study revealed the suitability of these glycomimetics for oral application in a proof-of-concept
infection model.
Results and Discussion
Design and synthesis of C-glycoside LecB inhibitors
We have previously modified the rather weak LecB ligand methyl α-D-mannoside (1) into
derivatives with various aromatic substituents attached to C-6, which increased potency up to a
factor of 20 and addressed a new subsite at the target (e.g., 2a-c and 3a-c, Figure 1, Table 1).30,32 In
contrast to rapidly dissociating 1 (t1/2 = 45 sec), these compounds showed extended receptor
residence times with complex half-lives in the 5-20 minute range, revealing the molecular basis for
the increased potency. This property is an important requirement for in vivo efficacy. In order to
further optimize target interactions and increase stability towards hydrolases, we merged functional
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groups establishing attractive interactions with LecB from the natural ligands into one molecule
(1→5, Figure 1).31 The equatorial methyl group in 5 resulted in C-glycosides with four- to sixfold
enhanced affinities originating from the reported lipophilic contact of the L-fucose methyl group
with the lectin.20,24,35 Despite these numerous improvements, the previously reported molecules
display only moderate affinities for LecB in the range of 3.3-16 µM, which is insufficient for an
application. Now, we aimed to combine the individually optimized structures of mannose-
derivatives 2a/3a and C-glycoside 5 into the monovalent drug-like molecules 6 and 7 to increase
affinity towards the target and assess their biological properties. Furthermore, these new
glycomimetic structures, 6 and 7, are expected to be more stable against host metabolism compared
to their glycosidic precursors, resulting from the absence of a labile glycosidic linkage. This design
should enable higher compound concentrations in vivo.
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Figure 1: Rational design of 6 and 7 as monovalent C-glycosidic glycomimetic LecB inhibitors. Derivatives of methyl
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α-D-mannoside 1-5 and their inhibitory potency for the binding with LecBPAO1
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Moieties colored in blue increase
potency by improving binding kinetics.30,32 The orange colored methyl group originating from L-fucosides enhances
binding to LecB through a lipophilic interaction.31,35
To access the β-C-glycosides 6 and 7, we utilized the previously described synthesis of amine 10,31
where L-fucose (8) was transformed to the β-C-glycoside 9 by Henry addition of nitromethane
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under basic conditions followed by a reduction to yield amine 10 (Scheme 1). Then, the
fucose/mannose hybrid-type structures 6 and 7 bearing amide or sulfonamide substituents,
respectively, were generated by coupling with different electrophiles.
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Besides the amide-bridged cinnamic acid moiety as present in 2a and 6a or the sulfonamide-bridged
trimethylphenyl moiety in 3a and 7a, a thiophenesulfonamide was introduced in mannoside 3b and
C-glycoside 7b as a bioisoster of the potent phenylsulfonamide 3c30 in the mannose-series. The final
coupling step yielded amides 6a,b and sulfonamides 7a,b in moderate to good yields (33-72%, over
2 steps).
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Scheme 1: Synthesis of the structures 6a,b and 7a,b. Reagents and conditions: (a) MeNO2, DBU, molecular sieves 3 Å,
1,4-dioxane, 50 °C, 3 d; (b) Pt/C, H2, HCl, MeOH, r.t., 2 d; (c) acyl/sulfonyl chloride or carboxylic acid/EDC*HCl,
Et3N, DMF, 0 °C; Yields are given over two steps from the nitro derivative 9.
Improved binding properties of C-glycoside structures towards LecB
Derivatives 6a,b and 7a,b were then analyzed for their capacity to inhibit the two representative
lectin variants of the two clinically occurring strain clades, LecBPAO1 and LecBPA14, based on
previously established competitive binding assays20,30 (Figure 2). Furthermore, all other
glycomimetics (i.e. 2a-c, 3a-c, 5) were assayed for the first time also towards LecBPA14 and showed
stronger binding to the PA14 variant compared to LecBPAO1, with the exception of cinnamides 2a,b.
In general, enhanced affinities were detected for C-glycoside derivatives 6a,b and 7a,b compared to
their respective mannose analogs 2a,b and 3a,b. Cinnamide 6a (IC50[LecBPAO1] 4.21 µM;
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IC50[LecBPA14] 2.49 µM) showed 9- to 16-fold higher affinities towards both LecB types compared
to its mannose reference 2a (IC50[LecBPAO1] 37 µM;30 IC50[LecBPA14] 39 µM). The sulfonamide 7a
(IC50[LecBPAO1] 0.97 µM; IC50[LecBPA14] 0.34 µM) was a 3- to 5-fold stronger ligand of LecB
compared to the corresponding mannoside 3a, exceeding the potency of the high-affinity natural
ligand L-fucose (8) for both lectin types (IC50[LecBPAO1] 2.74 µM;30 IC50[LecBPA14] 0.91 µM20).
Based on the four reference pairs analyzed, i.e., mannoside vs. C-glycoside: 2a/6a, 2b/6b, 3a/7a
and 3b/7b, which differ only at the anomeric position of mannose, a strong increase in affinity
towards LecB of up to 16-fold was identified for the C-glycosides. In general, the introduction of
amide and sulfonamide substituents always improved affinity for LecB compared to the
unsubstituted reference hybrid-structure 5.31 Importantly, for the first time the two clinically relevant
variants20 of LecB were analyzed with glycomimetic inhibitors, which showed potent inhibition for
both strain types.
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The thermodynamics of binding of derivatives 6a and 7a,b to both LecB variants was then studied
by isothermal titration calorimetry (Figure 3, Table S1-3). All ligands showed Kd values in the low
micro- to nanomolar range and a 1:1 binding stoichiometry, confirming the IC50 data obtained.
Notably, compared to the carbocyclic derivatives 6a and 7a (ΔH -37.8 to -31.4 kJ/mol), the
thiophene-containing ligand 7b showed an enhanced enthalpy driven binding (ΔH -50.0 and -48.1
kJ/mol), which was partially compensated by disfavored entropic contributions (-TΔS 13.4 and 12.7
kJ/mol). Thus, the introduced substituents also significantly enhanced binding enthalpy compared to
the unsubstituted congener 5 (ΔH -27.5 kJ/mol). An increased enthalpic contribution to binding was
previously introduced as a benchmark parameter assigned to a higher degree of target selectivity
due to a better quality of the drug-target interaction.36 Furthermore, physicochemical metrics such as
ligand efficiency and related ones, thought to gauge the quality of a drug-target interaction,37 are
promising. Importantly, the most potent compound thiophene 7b displayed enthalphy-driven
nanomolar affinities towards both LecB variants.
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Figure 2: Competitive binding assay of inhibitors with LecBPAO1 and LecBPA14. Means and standard deviations were
determined from a minimum of three independent measurements. IC50 values for 1 with both LecB variants and 2a-c,
3a,c and 5 with LecBPAO1 were previously published.20,30-32 One representative titration with LecBPA14 is depicted for the
reference pairs 2b/6b and 3a/7a. Grey arrows indicate the increase in activity for C-glycosides.
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Figure 3: Isothermal titration microcalorimetry of LecBPAO1 and LecBPA14 with ligands 6a, 7a,b. Means and standard
deviations were determined from a minimum of three independent titrations. One representative titration graph is
depicted for LecBPAO1 only.
We have previously detected a strong increase of the receptor half-lives for inhibitors 2a,b and 3a,
in comparison to natural carbohydrate ligand methyl α-D-mannoside (1).32 The role of ligand-
receptor binding kinetics is becoming increasingly significant for drug discovery and translation of
in vitro binding kinetics into cellular or in vivo effects was discussed.38 For the in vivo efficacy of a
drug molecule, prolonged receptor residence times are of importance. Ligand-receptor binding
kinetics of the C-glycosides 6a and 7a were studied by surface plasmon resonance (SPR) with
surface-bound LecBPAO1 (Figure 4). With its affinity of 330 nM obtained from SPR, the hybrid-type
structure 7a is a 140-fold more potent ligand of LecB than its carbohydrate ligand 1 and is tenfold
more potent than the natural high affinity ligand L-fucose (8) (see ITC experiments; Kd for 8 2.9
µM39). Most importantly, a further increase of the drug-receptor half lives for the C-glycosides 6a
and 7a over their mannose-based analogs 2a (t1/2 6.3 min and Kd by SPR 18.1 µM) and 3a (t1/2 18.6
min and Kd by SPR 1.12 µM)32 was observed. Receptor residence times of 28 minutes for 6a and 7a
resulted from very slow off rates of 4.1x10-4 s-1 of these C-glycosides. Thus, these glycomimetics
form a 38-fold more stable complex with the protein, compared to the natural ligand methyl α-D-
mannoside (1, t1/2 0.75 min, Kd by SPR 47.5 µM).32 These data validated the results from the
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competitive binding assay and ITC and further stress the impact of the additional equatorial methyl
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group in our glycomimetics.
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cmpd
Kd
[µM]
kon
[M-1s-1]
koff [s-
1]x103
t1/2
[min]
Rmax
[RU]
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7a
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Figure 4. Surface plasmon resonance (SPR) analysis of direct binding of 6a or 7a to immobilized LecBPAO1
.
Experimental data in black; calculated fits using a 1:1 binding model in red.
Structure of LecB in complex with hybrid inhibitors
To study the binding mode of the hybrid-type LecB inhibitors, we performed crystallization of these
ligands in complex with the lectin. Crystals of two ligands 7a and 7b in complex with LecBPA14
were obtained by the hanging drop co-crystallization method and the structures were solved to 1.65
Å resolution (Figure 5, Table S4). In both complexes, the carbohydrate-binding sites were occupied
with the respective inhibitors except one site in the LecB/7a complex.
Surprisingly, a slightly different binding mode for the methyl containing analog 7a was observed in
contrast to the previously reported structure30 of its mannose analog 2a (Figure 5A, Figures S1, S3).
While the carbohydrate-derived ring is in an identical position, the aromatic moiety attached to the
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exocyclic CH2 is rotated and located in a cleft of the protein surface that is enclosed by two
neighboring loops (S68-D75 and E95-D104) of the lectin. The methyl group attached to the
aromatic ring interacts with hydrophobic patches formed by V69 and a CH2-group of D96; one
sulfonamide oxygen of the ligand establishes a hydrogen bond with the backbone amide of G24. In
contrast for the mannoside 2a, a lipophilic contact between the protein surface (G24, V69) and the
mesitylene-ring was observed together with a hydrogen bond of its sulfonamide nitrogen with the
side chain of D96 (Figure S1). The latter interaction results from a conformational change of the
C5–C6 bond in the carbohydrate from a gt-orientation (N gauche to O5 and trans to O4), as present
in the complex of LecB with D-mannose (pdb code: for LecBPAO1 1OUR,40 or LecBPA14 5A6Y20), to a
tg-conformation in the mannoside 2a enabling this observed NH-D96 hydrogen bond. A
comparable binding mode as in the C-glycoside 7a was detected for its analog thiophene 7b (Figure
5B, Figures S1, S3). In the structure of mannoside 2a/LecB, a crystal lattice-dependent π-stacking
of the aromatic moiety with a second ligand from a neighboring LecB tetramer is observed with all
ligands in this structure showing the tg-conformation (Figure S2). Therefore, we consider the
binding modes reported here with the gt-orientation displayed by C-glycosides 7a and 7b as the
relevant ones, where crystal induced contacts are absent. In addition to the sulfonamide substituent
interactions, a lipophilic contact of the introduced C-glycosidic methyl group with the protein
residues T45 and A23 is observed in both analyzed complexes of hybrid-type ligands providing a
molecular basis for the measured affinity enhancement.
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Figure 5: Crystal structures of LecBPA14 with C-glycoside ligands: (A) Complex with trimethylphenyl sulfonamide 7a
(1.65 Å resolution), (B) Complex with thiophene 7b (1.65 Å resolution); For the ligands the 2Fobs–Fcalc electron density
is displayed at 1 σ. Ligands and amino acids of the carbohydrate recognition domain (CRD) are depicted as sticks
colored by elements (C: grey, N: blue, O: red, S: yellow); protein surface in transparent blue and two Ca2+-ions in the
binding sites are shown as green spheres.
Low molecular weight glycomimetics are potent inhibitors of bacterial biofilm formation
We then tested the most promising compounds in a P. aeruginosa biofilm assay. Most published
biofilm assays require staining and washing steps which impacted on the reproducibility in our
hands. Therefore, we generated genetically modified P. aeruginosa constitutively and intracellulary
expressing the red-fluorescent protein mCherry from the pMP7605 plasmid.41 Because fluorescence
intensity directly correlated with cell density (Figure S4), mCherry-derived fluorescence was
exploited as an internal stain for bacterial visualization and quantification of biofilm formation by
confocal laser scanning fluorescence microscopy. By eliminating washing and staining steps, this
method improved reproducibility through in situ imaging. A set of selected compounds comprising
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natural carbohydrates (mannoside 1, fucoside 11), glycomimetic mannose-derivatives (2a, 3a) and
C-glycosides (6a, 7a, 7b) was selected for assessment of their potential to inhibit biofilm formation
by P. aeruginosa.
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To exclude any unwanted antibiotic effect on biofilm formation, the total fluorescence intensity was
recorded after bacterial growth in presence of compounds for 23 h. Bacteria grew to the same
density as the DMSO control under each condition tested (Figure S5). Thus, these results clearly
indicated the absence of bactericidal or bacteriostatic effects for the tested compounds.
Figure 6: Inhibition of biofilm formation by P. aeruginosa after 48 h growth in presence of compounds 1, 2a, 3a, 6a,
7a, 7b or methyl α-L-fucoside (11) at 100 µM. DMSO in absence of compounds was used as control. (A) Quantification
of biofilm biomass. Averages and standard deviations of biofilm formation from three independent replicates. Statistical
significance was calculated using the students t-test. (B) Raw data of confocal fluorescence microscopy 3D images
show one representative z-stack per condition.
For inhibition of biofilm formation, culture medium was inoculated with P. aeruginosa
PA14/mCherry in presence of 100 µM compounds and culture and biofilm growth was allowed for
48 h, when biofilm mass was quantified using a confocal fluorescence microscope (Figure 6). All
analyzed compounds reduced biofilm formation by P. aeruginosa. Remarkably, the natural
carbohydrate ligands methyl α-D-mannoside (1) and methyl α-L-fucoside (11), showed only a small
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reduction in biofilm mass which was statistically insignificant. In contrast, all glycomimetics tested
showed a significant reduction of biofilm formation with the novel sulfonamide C-glycosides 7 as
most potent compounds reaching approx. 80% to 90% of inhibition. Mannose analogs 2a and 3a
also inhibited with a reduced potency the formation of biofilms with 54% and 66% observed
reduction, respectively.
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Compound selectivity for LecB over host lectins
Carbohydrate-binding proteins are ubiquitous and many play important roles in host innate immune
defense.42 Thus, inhibitors of LecB carrying natural carbohydrate epitopes may also result in
unspecific inhibition of human pathogen-recognition receptors. Langerin is a C-type lectin
expressed in human Langerhans cells and CD103+ dermal dendritic cells and recognizes
predominantly D-mannose, but also L-fucose, N-acetyl-D-glucosamine and sulfated
carbohydrates.43,44 As the C-glycosides presented here contain structural elements of D-mannose, L-
fucose and sulfonamide moieties, they are potential ligands of Langerin. Therefore, this protein was
chosen as one representative host lectin to study compound specificity. In order to assess the
compounds’ selectivity, 6a, 7a and its natural ligand L-fucose (8) were analyzed for Langerin
binding in a competitive 19F R2-filtered NMR assay44 (Figure 7). The natural ligand 8 was
recognized by Langerin with slightly decreased affinity (Ki of 7.2 mM) compared to D-mannose (Ki
4.5 mM44); C-glycosides 6a and 7a also showed moderate binding to this human protein in the
millimolar range. However, both compounds displayed a 1000-fold stronger affinity for LecB, and
are therefore considered selective for LecB over the host lectin Langerin.
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6a
7a
2.2 ± 0.6
8
Ki [mM]
4.0 ± 0.1
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Figure 7. Binding of C-glyocosides 6a, 7a and L-fucose (8) to Langerin as determined in a 19F R2-filtered NMR
competitive binding assay.
In addition to the direct binding to one host lectin, a more global effect of the glycomimetics on
immune homeostasis was investigated. Primary murine spleen cells were stimulated via different
pathways using bacterial lipopolysaccharide (LPS), fungal mannan or a combination of phorbol 12-
myristate 13-acetate (PMA) and ionomycin, and the immune response to these stimuli in presence
and absence of compounds 1, 2a, 3a, 6a, 7a, or 11 was monitored by quantifying TNF-α release
(Figure S6). No effect of mannoside 1 on the TNF-α levels was observed in comparison to matched
DMSO concentrations (Figure S6A), while cinnamide 2a showed a decrease in TNF-α levels
(Figure S6B). An immune suppressive effect was exclusively observed for cinnamide 2a at the
highest concentration tested (1 mM), while all other tested compounds, 3a, 6a, 7a and 11, showed
no effect on the immune response (Figure S6C). The immune-suppressive effect of 2a is an
interesting observation, however, the high concentration needed probably impedes further
development towards this effect. Excluding mannose-derived cinnamide 2a, none of the other
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compounds tested showed any effect on the immune response and provided further supporting
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In vitro metabolic stability and toxicity
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To achieve effective compound concentrations in vivo, efficient compound uptake and stability
towards host metabolism are crucial properties. Compound solubility is not considered problematic
for these well soluble carbohydrate derivatives. Calculated compound lipophilicity (clogP) showed
reasonable values (-1.25 < clogP < -0.13) for all compounds selected for early pharmacokinetics
and toxicity studies, i.e. sulfonamide C-glycosides 7a,b derivatives and the two mannose-based
compounds 3a and 3b (Table S5).
In vitro metabolic stability of the LecB inhibitors was studied against mouse and human liver
microsomes and murine plasma (Tables S6, S7, Figure S7). The data obtained revealed a low
intrinsic clearance (CLint) by mouse and human liver microsomes for all tested compounds. The
metabolic stability of the C-glycoside 7a (CLint 12 µL/min/mg protein) towards mouse microsomes
was slightly improved compared to its O-glycoside analog 3a (CLint 19 µL/min/mg protein)
supporting the initial rational design of these C-glycosides. In the second group, the stabilities of
mannoside 3b and C-glycoside 7b could not be differentiated since both compounds showed
highest stability in this assay. Remarkably, all tested compounds showed very low clearance by
human liver microsomes, where all substances fell into the highest stability category of this assay.
Stability in murine plasma was assessed and while the positive controls procaine and propoxycaine
were rapidly degraded, all analyzed LecB ligands were as stable as the negative control
procainamide and were not significantly degraded during the incubation period of two hours.
Therefore, all compounds tested can also be considered stable in murine plasma.
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In order to assess a first safety window, toxicity of compounds was studied in vitro using an
immortalized human hepatocyte cell line (Hep G2 cells, Figure S8). No toxicity was detected for all
compounds tested up to a concentration of 100 µM.
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In vivo pharmacokinetics of C-glycosides
The two candidates from the sulfonamides 7 with potent anti-biofilm effects and favorable ADME
properties, i.e. 7a and 7b, were chosen for an in vivo pharmacokinetics study in mice. Male CD-1
mice received a single dose of either 7a or 7b (10 mg/kg; n = 3) intravenously (i.v.) or perorally
(p.o.). Plasma and urine levels of the parent compounds were monitored during 24 h (Figure 8) and
pharmacokinetic parameters of 7a and 7b were assessed from plasma levels using a one-
compartment model (Table S10).45 In general, thiophene 7b had a superior plasma concentration
over mesitylene 7a achieving a two-fold higher calculated initial concentration (C0 13.4 µM for 7a
(i.v.); C0 28.3 µM for 7b (i.v.)) and thus both candidates exceeded the anticipated necessary
therapeutic levels (concentration > target Kd) with 10 mg/kg i.v.. Furthermore, 7b showed a slower
elimination rate (k10), a prolonged half-life (t1/2) and mean residence time (MRT) and a low
clearance (CL) compared to 7a. This resulted in higher compound levels in plasma at later time
points. These features also contribute to an overall drug exposure in plasma for 7b at an AUC of 7.4
µg*h/mL using the intravenous administration route, which was around fourfold higher than for 7a.
P.o. administration of 7a resulted in a bioavailability of nearly 100%, whereas for 7b it was only
around 33.5%. However, the p.o. route of administration of 7b still resulted in a higher AUC than
for 7a in the i.v. route. In addition, the p.o. route of 7b showed similar elimination curves as
detected for the i.v. route. In general, both compounds, 7a and 7b, were absorbed rapidly (tmax: 0.26-
0.51 h for 7a and 7b, p.o. route), which might be a consequence of the sugar-like structural
elements inherited by both compounds facilitating the crossing of barriers. As unmetabolized 7b
was detected in urine at high concentrations (Figure 8), its elimination from the systemic circulation
likely proceeds via renal clearance rather than liver detoxification. The kidneys did not act as a
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reservoir and thiophene 7b did not accumulate in this organ since its concentration in kidney 24 h
post i.v. dosing was below 6 nM. Taken together, both compounds showed good oral availability
and thiophene 7b showed a superior pharmacokinetic profile compared to 7a with high compound
levels, both in plasma and urine.
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Figure 8: In vivo pharmacokinetics of 7a and 7b in CD-1 mice. Mean profile (±) SD of plasma (left column) and urine
(right column) concentration in mouse versus time after i.v. (top) or p.o. (bottom) administration of compound 7a or 7b
in a single dosing experiment (10 mg/kg, n=3). Dashed line represents the in vitro IC50 range for both candidates with
LecBPA14
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Conclusions
We have developed the first low molecular weight LecB inhibitors that potently inhibited biofilm
formation by P. aeruginosa, one of the prime mechanisms of antimicrobial resistance of this
pathogen of outstanding medical importance. The synthesized glycomimetic C-glycosides showed
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inhibitory affinities as low as nanomolar for LecB variants from two strains representative for the
entire set of clinical isolates.20 Thus, it can be expected that the devised glycomimetics will be
active against LecB-mediated biofilm formation in a broad spectrum of clinical isolates.
The compounds showed excellent biophysical properties with receptor residence times as high as 28
minutes and fully enthalpy driven binding to the target lectin, which are two important benchmarks
for early drug development. In contrast to the ineffective natural carbohydrate ligands, our
glycomimetics were potent inhibitors of biofilm formation without affecting bacterial viability.
Thus, development of resistances towards these pure anti-biofilm substances is likely to be reduced,
in contrast to this well-known problem for traditional antibiotics.12,46
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Target selectivity was then studied in binding experiments towards Langerin, a human fucose- and
mannose-binding C-type lectin, as well as in a more global approach in immune-stimulated primary
murine spleen cells and no impeding effects of the glycomimetics could be observed. All
compounds tested showed absence of toxicity. Furthermore, a set of in vitro ADME experiments
revealed good metabolic stability of the compounds against liver microsomes, murine and human
plasma and a balanced lipophilicity that is important for oral availability.
In a first in vivo experiment, the pharmacokinetics of 7a and 7b in mice revealed oral availability
with high plasma concentrations and a subsequent primary excretion route via the kidneys.
Consequently, high compound concentrations in urine were observed, suggesting but not limiting to
an application in a P. aeruginosa urinary tract infection (UTI) model. It will now be of interest to
evaluate the compounds’ anti-infective potential in a mono-therapy treatment against biofilm-
associated infections and their synergy together with antibiotics for efficient eradication of bacteria
outside the biofilm.
Associated Content
The Supporting Information is available free of charge on the ACS Publications website.
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Supporting Information contain experimental details and H and C spectra of new compounds;
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ITC titration data; X-ray Data collection and refinement statistics; Crystal Structures showing
ligand alignment and crystal packing effects; Correlation of fluorescence intensities with cfu and
OD600 measurements; Effects of compounds on total fluorescence intensities; Calculated
lipophilicity of selected compounds; Analysis of TNF-α concentration after stimulation of mouse
spleen cells with and without test compounds; Microsomal intrinsic clearance (CLint) of 3a, 3b,
and C-glycosides 7 in mouse and human liver microsomes; Stability of LecB ligands in mouse
plasma; m/z search window for plasma stability assay; Toxicity of LecB ligands to human liver Hep
G2 cells; Accuracy, quantification limits and lower limit of qualification for 7a and 7b in plasma,
urine and kidney matrix; Mass spectrometric conditions used for quantification and qualification of
7a, 7b and the internal standard glipizide; PK parameters of 7a and 7b in mice.
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Author Information
Corresponding Author
* alexander.titz@helmholtz-hzi.de
ORCID
Roman Sommer 0000-0003-0592-0810
Annabelle Varrot 0000-0001-6667-8162
Eike-Christian Wamhoff 0000-0002-8665-5512
Christoph Rademacher 0000-0001-7082-7239
Rolf W. Hartmann 0000-0002-5871-5231
Mark Brönstrup 0000-0002-8971-7045
Anne Imberty 0000-0001-6825-9527
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Alexander Titz 0000-0001-7408-5084
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Notes
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The authors declare no competing financial interest.
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Acknowledgments
The authors are grateful to Sarah Henrikus and Shelby Newsad for chemistry support and to Dr.
Michael Hoffmann for HRMS measurements (all HIPS Saarbrücken); we acknowledge technical
assistance from Tatjana Arnold (HZI, Braunschweig) and Astrid Glöckner (Konstanz University);
Dr. Aymeric Audfray (CERMAV Grenoble) is acknowledged for instructions to SPR and Dr. Josef
Zapp (Saarland University) for performing NMR measurements. Crystal data collection was
performed at the European Synchrotron Radiation Facility, Grenoble, France and we are grateful for
access and technical support to beamline FIP-BM30A and ID29. A.I. and A.V. acknowledge
support from the ANR projects Glyco@Alps (ANR-15-IDEX-02) and Labex ARCANE (ANR-11-
LABX-003). We thank the Helmholtz Association (grant no VH-NG-934, to A.T.), EU COST
action BM1003 (to R. S.), DAAD RISE program (to S. Newsad and R.S.) and the Deutsche
Forschungsgemeinschaft (to A.T., grant no Ti756/2-1, to C.R., grant no RA1944/2-1) for financial
support.
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Bibliography
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1. Nagao, M.; Iinuma, Y.; Igawa, J.; Saito, T.; Yamashita, K.; Kondo, T.; Matsushima, A.;
Takakura, S.; Takaori-Kondo, A.; Ichiyama, S. J Hosp Infect 2011, 79, 49-53.
8
9
2. Rice, L. B. J Infect Dis 2008, 197, 1079-1081.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Tsutsui, A.; Suzuki, S.; Yamane, K.; Matsui, M.; Konda, T.; Marui, E.; Takahashi, K.;
Arakawa, Y. J Hosp Infect 2011, 78, 317-22.
4. World Health Organization 2017. Available at:
http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/. Accessed
May 2017.
5. Hauser AR, Rello J. Severe Infections Caused by Pseudomonas Aeruginosa. Kluwer Academic
Publishers Group; 2003.
6. Serra, R.; Grande, R.; Butrico, L.; Rossi, A.; Settimio, U. F.; Caroleo, B.; Amato, B.; Gallelli,
L.; de Franciscis, S. Expert Rev Anti Infect Ther 2015, 13, 605-13.
7. Mittal, R.; Aggarwal, S.; Sharma, S.; Chhibber, S.; Harjai, K. J Infect Public Health 2009, 2,
101-11.
8. Poole, K. Front Microbiol 2011, 2, 65.
9. Flemming, H.-C.; Wingender, J. Nat Rev Microbiol 2010, 8, 623-33.
10. Davies, D. Nat Rev Drug Discov 2003, 2, 114-22.
11. Sommer, R.; Joachim, I.; Wagner, S.; Titz, A. CHIMIA 2013, 67, 286-90.
12. Wagner, S.; Sommer, R.; Hinsberger, S.; Lu, C.; Hartmann, R. W.; Empting, M.; Titz, A. J Med
Chem 2016, 59, 5929-5969.
13. Winzer, K.; Falconer, C.; Garber, N. C.; Diggle, S. P.; Camara, M.; Williams, P. J Bacteriol
2000, 182, 6401-11.
14. Diggle, S. P.; Stacey, R. E.; Dodd, C.; Cámara, M.; Williams, P.; Winzer, K. Environ Microbiol
2006, 8, 1095-104.
15. Tielker, D.; Hacker, S.; Loris, R.; Strathmann, M.; Wingender, J.; Wilhelm, S.; Rosenau, F.;
Jaeger, K.-E. Microbiology 2005, 151, 1313-23.
16. Gilboa-Garber, N. Methods Enzymol 1982, 83, 378-385.
17. Klockgether, J.; Cramer, N.; Wiehlmann, L.; Davenport, C. F.; Tümmler, B. Front Microbiol
2011, 2, 150.
18. Dötsch, A.; Schniederjans, M.; Khaledi, A.; Hornischer, K.; Schulz, S.; Bielecka, A.; Eckweiler,
D.; Pohl, S.; Häussler, S. MBio 2015, 6, e00749-15.
19. Boukerb, A. M.; Decor, A.; Ribun, S.; Tabaroni, R.; Rousset, A.; Commin, L.; Buff, S.;
Doléans-Jordheim, A.; Vidal, S.; Varrot, A.; Imberty, A.; Cournoyer, B. Front Microbiol 2016,
7, 811.
20. Sommer, R.; Wagner, S.; Varrot, A.; Nycholat, C. M.; Khaledi, A.; Haussler, S.; Paulson, J. C.;
Imberty, A.; Titz, A. Chem. Sci. 2016, 7, 4990-5001.
21. Boukerb, A. M.; Rousset, A.; Galanos, N.; Méar, J.-B.; Thepaut, M.; Grandjean, T.; Gillon, E.;
Cecioni, S.; Abderrahmen, C.; Faure, K.; Redelberger, D.; Kipnis, E.; Dessein, R.; Havet, S.;
Darblade, B.; Matthews, S. E.; de Bentzmann, S.; Guéry, B.; Cournoyer, B.; Imberty, A.; Vidal,
S. J Med Chem 2014, 57, 10275-10289.
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5
22. Chemani, C.; Imberty, A.; de Bentzmann, S.; Pierre, M.; Wimmerová, M.; Guery, B. P.; Faure,
K. Infect Immun 2009, 77, 2065-75.
23. Cott, C.; Thuenauer, R.; Landi, A.; Kühn, K.; Juillot, S.; Imberty, A.; Madl, J.; Eierhoff, T.;
Römer, W. Biochim Biophys Acta 2016, 1863, 1106-18.
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7
8
9
24. Mitchell, E.; Houles, C.; Sudakevitz, D.; Wimmerova, M.; Gautier, C.; Pérez, S.; Wu, A. M.;
Gilboa-Garber, N.; Imberty, A. Nat Struct Biol 2002, 9, 918-21.
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25. Hauber, H.-P.; Schulz, M.; Pforte, A.; Mack, D.; Zabel, P.; Schumacher, U. Int J Med Sci 2008,
5, 371-6.
26. Bucior, I.; Abbott, J.; Song, Y.; Matthay, M. A.; Engel, J. N. Am J Physiol Lung Cell Mol
Physiol 2013, 305, L352-63.
27. Cecioni, S.; Imberty, A.; Vidal, S. Chem Rev 2015, 115, 525-61.
28. Bernardi, A.; Jiménez-Barbero, J.; Casnati, A.; De Castro, C.; Darbre, T.; Fieschi, F.; Finne, J.;
Funken, H.; Jaeger, K.-E.; Lahmann, M.; Lindhorst, T. K.; Marradi, M.; Messner, P.; Molinaro,
A.; Murphy, P. V.; Nativi, C.; Oscarson, S.; Penadés, S.; Peri, F.; Pieters, R. J.; Renaudet, O.;
Reymond, J.-L.; Richichi, B.; Rojo, J.; Sansone, F.; Schäffer, C.; Turnbull, W. B.; Velasco-
Torrijos, T.; Vidal, S.; Vincent, S.; Wennekes, T.; Zuilhof, H.; Imberty, A. Chem Soc Rev 2012,
42, 4709-4727.
29. Johansson, E. M. V.; Crusz, S. A.; Kolomiets, E.; Buts, L.; Kadam, R. U.; Cacciarini, M.;
Bartels, K.-M.; Diggle, S. P.; Cámara, M.; Williams, P.; Loris, R.; Nativi, C.; Rosenau, F.;
Jaeger, K.-E.; Darbre, T.; Reymond, J.-L. Chem Biol 2008, 15, 1249-57.
30. Hauck, D.; Joachim, I.; Frommeyer, B.; Varrot, A.; Philipp, B.; Möller, H. M.; Imberty, A.;
Exner, T. E.; Titz, A. ACS Chem Biol 2013, 8, 1775-84.
31. Sommer, R.; Exner, T. E.; Titz, A. PLoS One 2014, 9, e112822.
32. Sommer, R.; Hauck, D.; Varrot, A.; Wagner, S.; Audfray, A.; Prestel, A.; Möller, H. M.;
Imberty, A.; Titz, A. ChemistryOpen 2015, 4, 756-767.
33. Hofmann, A.; Sommer, R.; Hauck, D.; Stifel, J.; Göttker-Schnetmann, I.; Titz, A. Carbohydr
Res 2015, 412, 34-42.
34. Beshr, G.; Sommer, R.; Hauck, D.; Siebert, D. C. B.; Hofmann, A.; Imberty, A.; Titz, A. Med
Chem Commun 2016, 7, 519-530.
35. Sabin, C.; Mitchell, E. P.; Pokorná, M.; Gautier, C.; Utille, J.-P.; Wimmerová, M.; Imberty, A.
FEBS Lett 2006, 580, 982-7.
36. Tarcsay, A.; Keseru, G. M. Drug Discovery Today 2015, 20, 86-94.
37. Hopkins, A. L.; Keseru, G. M.; Leeson, P. D.; Rees, D. C.; Reynolds, C. H. Nat Rev Drug
Discov 2014, 13, 105-21.
38. Tummino, P. J.; Copeland, R. A. Biochemistry 2008, 47, 5481-92.
39. Perret, S.; Sabin, C.; Dumon, C.; Pokorná, M.; Gautier, C.; Galanina, O.; Ilia, S.; Bovin, N.;
Nicaise, M.; Desmadril, M.; Gilboa-Garber, N.; Wimmerová, M.; Mitchell, E. P.; Imberty, A.
Biochem J 2005, 389, 325-32.
40. Loris, R.; Tielker, D.; Jaeger, K.-E.; Wyns, L. J Mol Biol 2003, 331, 861-70.
41. Lagendijk, E. L.; Validov, S.; Lamers, G. E. M.; de Weert, S.; Bloemberg, G. V. FEMS
Microbiol Lett 2010, 305, 81-90.
42. Mayer, S.; Raulf, M.-K.; Lepenies, B. Histochem Cell Biol 2017, 147, 223-237.
43. Holla, A.; Skerra, A. Protein Eng Des Sel 2011, 24, 659-69.
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44. Wamhoff, E.-C.; Hanske, J.; Schnirch, L.; Aretz, J.; Grube, M.; Varón Silva, D.; Rademacher,
C. ACS Chem Biol 2016, 11, 2407-13.
45. Zhang, Y.; Huo, M.; Zhou, J.; Xie, S. Comput Methods Programs Biomed 2010, 99, 306-14.
46. Allen, R. C.; Popat, R.; Diggle, S. P.; Brown, S. P. Nat Rev Microbiol 2014, 12, 300-8.
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