Cinnamide Derivatives of d-Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

Article abstract of DOI:10.1002/open.201500162

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen.

Full text of DOI:10.1002/open.201500162

DOI: 10.1002/open.201500162
Cinnamide Derivatives of d-Mannose as Inhibitors of the
Bacterial Virulence Factor LecB from Pseudomonas
aeruginosa**
Roman Sommer,[a, b, c] Dirk Hauck,[a, b, c] Annabelle Varrot,[d] Stefanie Wagner,[a, c]
Aymeric Audfray,[d] Andreas Prestel,[b, e] Heiko M. Mçller,[b, e] Anne Imberty,[d] and
Alexander Titz*[a, b, c]
Pseudomonas aeruginosa is an opportunistic Gram-negative
pathogen with high antibiotic resistance. Its lectin LecB was
identified as a virulence factor and is relevant in bacterial ad-
hesion and biofilm formation. Inhibition of LecB with carbohy-
drate-based ligands results in a decrease in toxicity and biofilm
formation. We recently discovered two classes of potent drug-
like glycomimetic inhibitors, that is, sulfonamides and cinna-
mides of d-mannose. Here, we describe the chemical synthesis
and biochemical evaluation of more than 20 derivatives with
increased potency compared to the unsubstituted cinnamide.
The structure–activity relationship (SAR) obtained and the ex-
tended biophysical characterization allowed the experimental
determination of the binding mode of these cinnamides with
LecB. The established surface binding mode now allows future
rational structure-based drug design. Importantly, all glycomi-
metics tested showed extended receptor residence times with
half-lives in the 5–20 min range, a prerequisite for therapeutic
application. Thus, the glycomimetics described here provide an
excellent basis for future development of anti-infectives
against this multidrug-resistant pathogen.
Introduction
The Gram-negative and opportunistic pathogen Pseudomonas
aeruginosa is a major threat to hospitalized and immune-com-
promised patients.[1] Unfortunately, many hospital strains are
getting largely unaffected by many antibiotics.[2] This resistance
results from the presence of drug-neutralizing tools in P. aeru-
ginosa, for example, beta-lactamases or efflux pumps, but also
from its potential to form biofilms. In these biofilms, bacteria
are embedded in a self-produced matrix, which protects
against host immune defense and antibiotic treatment.[3,4]
Two lectins produced by P. aeruginosa, LecA (PA-IL) and LecB
(PA-IIL), play key roles in biofilm formation, and both lectins
are virulence factors involved in host damage and bacterial
uptake.[5–10] Inhibition of these lectins as an anti-infective strat-
egy belongs to the new concept of antivirulence therapies.[11]
In contrast to antibiotics targeting bacterial viability, antiviru-
lence therapeutics solely target the infection without interfer-
ence on viability and therefore the appearance of resistant mu-
tants is decreased.[12] In a proof of concept study for therapeu-
tic suitability of the lectins LecA and LecB as target, Hauber
et al. could show a decrease in bacterial load after treatment
of pulmonary-infected patients with an aerosol containing nat-
ural ligands of both lectins.[13]
[a] R. Sommer, D. Hauck, Dr. S. Wagner, Dr. A. Titz
Chemical Biology of Carbohydrates
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS)
Universitätsstrasse 10, 66123 Saarbrücken (Germany)
E-mail: alexander.titz@helmholtz-hzi.de
[b] R. Sommer, D. Hauck, A. Prestel, Prof. H. M. Mçller, Dr. A. Titz
Department of Chemistry and Graduate School Chemical Biology
University of Konstanz, 78457 Konstanz (Germany)
[c] R. Sommer, D. Hauck, Dr. S. Wagner, Dr. A. Titz
Deutsches Zentrum für Infektionsforschung (DZIF)
Inhoffenstraße 7, 38124 Braunschweig (Germany)
[d] Dr. A. Varrot, Dr. A. Audfray, Dr. A. Imberty
Centre de Recherche sur les MacromolØcules VØgØtales (CERMAV-UPR5301)
CNRS and UniversitØ Grenoble Alpes
The carbohydrate-binding protein LecB was first isolated by
Gilboa–Garber et al., and its glycan preference was deter-
mined.[14,15] The crystal structure of LecB in complex with its
high-affinity ligand l-fucose revealed the presence of two calci-
um ions, which mediate binding between the saccharide and
the protein.[16] A subsequent study could show that the lower-
affinity ligand d-mannose binds similarly to LecB as a conse-
quence of three hydroxy groups with identical relative orienta-
tion in both l-fucose and d-mannose.[17] The trisaccharide
Lewisa was identified from glycan array analysis and is currently
its best known monovalent ligand with a thermodynamic dis-
sociation constant of 210 nm.[18,19] Numerous multivalent inhib-
BP53, 38041 Grenoble cedex 9 (France)
[e] A. Prestel, Prof. H. M. Mçller
Institute of Chemistry, University of Potsdam, 14476 Potsdam (Germany)
[**] This article is part of the Virtual Special Issue “Carbohydrates in the 21st
Century: Synthesis and Applications”.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201500162.
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
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Ser23 was observed, particular attention was paid to this hy-
droxy group in the design of new inhibitors.[25,27,28] However,
we could show through chemical modification that this hydro-
gen bond does not have a significant influence on binding af-
finity at ambient conditions in aqueous solution.[27,28] In our
previous study, we succeeded in obtaining a co-crystal struc-
ture of sulfonamide 2 in complex with LecB. In the absence of
a crystal structure for cinnamide 3 in complex with LecB, mo-
lecular dynamics simulations and NMR suggested an intercala-
tion of the cinnamide residue into the beta-sandwich of the
lectin.[25]
Here, we describe the synthesis of a set of more than 20
substituted cinnamido derivatives to establish the structure–
activity relationship (SAR) of the interaction of this class of in-
hibitors with LecB. Furthermore, we used a diverse set of dif-
ferent biophysical techniques to establish the binding mode of
this class of LecB inhibitors.
Figure 1. Reported natural and synthetically modified mannose based inhibi-
tors 1, 2, and 3 and their thermodynamic dissociation constants (Kd)with the
lectin LecB.[25,26]
itors of LecB based on conjugates of natural l-fucose have
been reported to date in order to further increase binding af-
finities.[20,21] One multivalent fucosylated peptide dendrimer
was shown to be a high-affinity ligand with the potential to in-
hibit biofilm formation and disperse established biofilms of
P. aeruginosa in a LecB-dependent fashion.[22] In contrast, the
development of monovalent inhibitors has been largely ne-
glected due to their intrinsically lower affinities and efforts are
summarized in recent reviews.[23,24]
Results and Discussion
In the predicted binding mode of cinnamide 3, the aromatic
moiety opens a cleft in the protein and intercalates into the
beta-sandwich formed by one LecB monomer. In order to get
a more detailed picture of the interaction of such cinnamides
with LecB, we performed a structure–activity study with a varie-
ty of substituents in ortho-, meta-, and para-position of the cin-
namic amide residue of 3. The synthesis of the cinnamide de-
rivatives started from methyl a-d-mannoside (1). Introduction
of an amino functional group allowed amide bond formation
with unprotected mannose derivative 4 with a set of selected
commercially available cinnamic acid derivatives. Various sub-
stituents were introduced in ortho-, meta-, or para-position re-
sulting in the potential LecB inhibitors 5a–e, 5g–k, and 5m–q,
respectively, which were obtained in moderate to good yields
(Scheme 1). The nitro derivatives 5e, 5k, and 5q were further
We recently reported the discovery of derivatives of d-man-
nose with amido- and sulfonamido substituents at position 6
(2 and 3, Figure 1).[25] Compared with the parent low-affinity
ligand methyl mannoside (1, Figure 1),[26] these modifications
improved binding affinity between four- and 24-fold. In con-
trast to a-l-fucosides, d-mannose as scaffold provided the pos-
sibility to target an adjacent cleft on the protein in order to in-
crease binding affinity. Because in the crystal structure of LecB
with d-mannose,[17] a hydrogen bond between its 6-OH and
Scheme 1. Synthesis of compounds 5a–r, 6, and 7a,b. Reagents and conditions: a) carboxylic acids, EDC·HCl, Et3N, DMF, 08C ! rt; b) FeSO4·7H2O, NH4OH,
H2O, rt; c) Pd/C, H2, MeOH, rt. Reaction times, yields, and purities are summarized in Table S1 in the Supporting Information.
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reduced with iron(II) sulfate following a method by Pribulova
et al.[29] for aliphatic nitro groups to give the corresponding
anilines 5 f, 5l, and 5r in high yields. Furthermore, we synthe-
sized the more rigid 2-naphtoyl derivative 6 as a cyclized
analog of 3, as well as the two disubstituted analogs 7a and
7b following the same protocol.
All synthesized compounds were then biochemically evalu-
ated for inhibition of LecB using a competitive binding assay
based on fluorescence polarization readout, recently devel-
oped for LecB in our lab.[25] Surprisingly, all monosubstituted
compounds were within a narrow activity range with IC50
values from 27–73 mm, indicating that the substituents tested
had a moderate effect on the activity that remains similar to
the parent cinnamide 3 with an IC50 of 37 mm (Figure 2). Intro-
duction of ortho substituents in 5a–f generally resulted in
weaker inhibition of LecB (IC50 49–73 mm), while the same sub-
stituents in meta- or para-position (5g–l and 5m–r) showed
similar or enhanced potency with IC50 values ranging from 27–
53 mm. The decreased affinity of ortho-substituted 5a–f might
result from a steric hindrance
sulted in enhanced potencies especially in meta or para posi-
tion (5g, 5i, 5j, and 5m, 5o, 5p). Methoxy-substituted cinna-
mides were superior inhibitors in the ortho, meta, and para
series, with highest potencies for meta-5i and para-5o. We
therefore combined both substituents in one molecule as 3,4-
methylenedioxy derivative 7a or 3,4-dimethoxycinnamide 7b
to analyze potential synergistic effects. Both compounds
showed good inhibition of LecB with dimethoxy-substituted
7b as the most potent inhibitor at an IC50 of 19.9 mm. Methyle-
nedioxy derivative 7a had an IC50 of 29 mm, which was compa-
rable to singly meta- or para-methoxy-substituted 5i and 5o.
In addition, we tested the cyclized analog 6 bearing a 2-
naphtamide in lieu of the cinnamide moiety for LecB inhibi-
tion. In accordance with the general trend of higher affinities
for more lipophilic substituents among all tested compounds,
compound 6 also displayed an increased inhibitory potency
(IC50 31 mm). The 2-naphtamide 6 is structurally similar to
ortho-substituted cinnamides 5a–f, especially to the 2-methyl
derivative 5d. Generally, steric hindrance with the receptor,
with the receptor. Generally, the
introduction of polar substitu-
ents such as phenolic hydroxy
groups or aniline-NH2 led to a de-
creased affinity (5b, 5 f, 5h, 5l,
5n, 5r), whereas the presence of
the
lipophilic
substituents
Scheme 2. Synthesis of glucose-derivative 10. Reagents and conditions: a) Pd/C, H2, MeOH, rt; b) cinnamic acid,
chloro, methoxy, or methyl re- EDC·HCl, Et3N, DMF, 08C ! rt.
Figure 2. Biochemical evaluation of LecB binding to ligands 5a–r, 6, and 7a,b. IC50 values were determined with a competitive fluorescence polarization
assay. Means and standard deviations were determined from a minimum of three independent experiments.
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a distorted planarity of the cinnamide moiety, or even a further
increase of the already high rotational barrier due to the ortho-
substituents in 5a–f could account for the decreased affinity in
the ortho-series. Possible reasons for the difference in activity
between 6 and 5a–f cannot be deduced from the present
data set. Although a rather flat SAR on the substituents of the
cinnamic acid moiety was obtained, a two-fold increase in po-
tency compared to unsubstituted cinnamide 3 could be ach-
ieved through introduction of two methoxy substituents in
meta- and para position in 7b.
Can this SAR at the cinnamide residue explain the previously
proposed intercalation model of the binding of 3 to LecB?
During our previous structural characterization of the cinna-
mide 3–LecB interaction, we observed global line broadening
of the protein signals at a protein–ligand ratio of 2:1; a stoi-
chiometric ratio of 1:1 led to complete vanishing of resonances
and visual precipitation of protein after a few hours of record-
ing time. In contrast, the same analysis with sulfonamide 2
yielded distinct shifts of a small set of protein resonances. The
protein NMR spectra were interpreted, such that the computa-
tionally predicted intercalation of 3 into the beta-sandwich of
LecB leads to a global effect on its structure and, thus, results
in changes of most of the protein resonances.
Figure 3. Inhibition of LecB by manno-3 is carbohydrate-specific, and the
control compound gluco-10 has no effect up to a concentration of 3 mm on
LecB functional binding to the fucosylated fluorescent probe of the assay
system.
In order to analyze whether the observed effects on LecB in-
duced by cinnamide 3 are carbohydrate-specific and to ex-
clude simple detergent-like denaturation of the protein, we de-
signed its glucose-analog 10. The compound was synthesized
in analogy to 3: methyl a-d-glucoside (8) was transformed into
the tosylate and subsequently the azide 9 was obtained after
sodium azide treatment as previously reported by Cramer
et al.[30] (Scheme 2). The azide was hydrogenolytically reduced
using palladium on activated charcoal, and the resulting amine
was directly coupled with cinnamic acid to yield gluco-
cinnamide 10 in good yield (46% over two steps).
The control compound gluco-10 was then tested in the com-
petitive binding assay to assess the carbohydrate-based specif-
icity of cinnamide manno-3. No inhibition of LecB function up
to 3 mm of gluco-cinnamide 10 was observed (Figure 3),
whereas manno-3 showed inhibition in a dose-dependent
manner as previously reported. This assay relies on the dis-
placement of a fucose-fluorescein conjugate as detection
probe bound to intact LecB. The observed high fluorescence
polarization even at high concentrations of gluco-10 therefore
indicated persistent binding of the fluorescent probe and thus
the absence of any kind of inhibitory influence of gluco-10 on
LecB function. In summary, there was no unspecific binding of
the cinnamide moiety at elevated ligand concentrations, but
the effects observed with manno-3 were instead dependent
on the stereochemistry of one single hydroxy group and thus
on the specific binding to the carbohydrate recognition site of
LecB.
Figure 4. Isothermal titration microcalorimetry (ITC) of LecB with dimethoxy-
cinnamide 7b. Three independent titrations were performed; one represen-
tative example is shown here.
To further study the interaction of the most potent cinna-
mide with LecB, compound 7b was then titrated to LecB, and
the thermodynamics of binding were analyzed by isothermal
microcalorimetry (ITC, Figure 4, Table 1). A Kd value of 10.9 mm
with a stoichiometry of 1 was determined, and binding enthal-
py and entropy were obtained. In comparison to sulfona-
mide 2, both cinnamide 3 and dimethoxycinnamide 7b
showed a reduced binding enthalpy, but in contrast to 2,
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was threefold slower than mannoside 1 and more than fivefold
slower than sulfonamide 2. Its dimethoxy derivative 7b also
Table 1. Thermodynamics of ligand binding to LecB by isothermal titra-
tion microcalorimetry. Values for 1–3 were taken from the literature.[25,26]
showed a slow on rate with a kon of 169.6mÀ1 À1, indicating
s
7b
3
2
MeMan [1]
a similar mode of binding to LecB for 3 and 7b. The dissocia-
tion constant of 18.1 mm for 3 was in very good agreement
with its previously determined value of 18.5 mm[25] by calorime-
try. The most potent cinnamide derivative dimethoxycinnami-
de 7b showed a Kd by SPR of 7.7 mm and a complex half-life t1/
Kd [mm]
10.9Æ1.80
À6.81Æ0.16
À5.63Æ0.21
À1.18Æ0.36
1.03Æ0.09
18.5
À6.6
À4.3
À2.3
3.3
À7.5
À7.9
0.4
71
DG [kcalmolÀ1
DH [kcalmolÀ1
]
]
À5.4
À4.3
À1.4
0.94
ÀTDS [kcalmolÀ1
]
N
0.94
0.71
of 8.8 min, demonstrating the high potential of this series for
2
further drug development. The very slow on rates observed for
the cinnamides 3 and 7b could result from a conformational
change within the protein upon binding, in favor of the previ-
ously proposed intercalation model or from conformational
changes within the ligand.
a beneficial contribution of binding entropy to the Gibbs free
energy.
Subsequently, we performed surface plasmon resonance
(SPR) analysis to determine the parameters of binding kinetics
of LecB with its ligands. LecB
was biotinylated using an acti-
vated carboxylic acid derivative
of biotin and coupled to the sur-
face of
a streptavidin-coated
sensor chip. High surface densi-
ties of LecB were obtained,
which is necessary for the detec-
tion of small molecule binding.
Methyl mannoside (1), sulfona-
mide 2, cinnamide 3, and its de-
rivative 7b, which showed the
highest potency among the cin-
namide series in LecB inhibition,
were analyzed by SPR (Figure 5,
Table 2). Unmodified manno-
side 1 showed fast binding ki-
netics, which are usually ob-
served with lectin–carbohydrate
interactions.[31] The measured Kd
of 47 mm was in good agree-
ment with the previously deter-
mined thermodynamic value of
71 mm by ITC.[26] Sulfonamide 2
showed a two-fold higher on
rate and a very slow off rate of
6.2”10À4 sÀ1. The half-life (t1/2
=
ln2/koff) of the complex of LecB
and sulfonamide 2 was 18.6 min,
explaining its very high affinity
(Kd =1.12 mm by SPR, 3.3 mm[25]
by ITC) towards LecB compared
to a t1/2 of only 45 s for 1. It is
important to note, that long
drug-receptor half lives are im-
portant for in vivo function and
future success for drug develop-
ment.[32,33] When analyzing cin-
namide 3, slow binding kinetics
approaching the detection limit
of the apparatus were observed
Figure 5. Single cycle kinetics analysis by surface plasmon resonance (SPR) of the direct binding of 1, 2, 3, or 7b
to immobilized LecB. Experimental data are shown in black; calculated fits using a 1:1 binding model with
a global fitting on all injected concentrations are shown in red. Ligand concentrations injected were 120 nm,
with a kon of 100.9mÀ1 sÀ1, which 600 nm, 3 mm, 15 mm, and 75 mm for 2, 3, and 7b, and 3 mm, 15 mm, 75 mm, 375 mm, and 1.8 mm for 1.
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protein (data not shown). The lack of success in both thermal
unfolding techniques presumably originates in the extraordina-
ry high thermal stability of LecB.[14]
Table 2. Data obtained from the kinetic fit of a 1:1 binding model to the
SPR single cycle kinetics sensograms.
cmpd Kd [mM] kon [mÀ1 sÀ1
]
koff [sÀ1
]
koff [sÀ1]”103 t1/2 [min] Rmax
Based on the high degree of structural similarity of cinna-
mide 3 and dimethoxycinnamide 7b and their similar binding
kinetics, we anticipated comparable binding modes and thus
also a similar behavior on protein NMR signals. Surprisingly,
1
2
3
47.5
1.12
18.1
7.67
326.1
552.6
100.9
169.6
0.01548
0.00062
0.00183
0.00130
15.48
0.62
1.83
1.30
0.75
18.64
6.33
18.8
94.9
93.8
7b
8.88
107.0
1
the H,15N-TROSY-HSQC (transverse relaxation-optimized spec-
troscopy–heteronuclear single quantum coherence) NMR spec-
trum of 15N-labelled LecB showed only distinct shifts of protein
resonances in the presence of 7b, and even saturation of LecB
with dimethoxycinnamide 7b could be achieved at excellent
quality of the spectra (Figure 7B). In contrast to our previously
reported data for LecB in complex with the unsubstituted cin-
namide 3, no global vanishing of signal intensity was observed
with dimethoxycinnamide 7b. The behavior observed with 7b
rather resembles the spectra obtained with sulfonamide 2 and
thus a distinctly defined binding site.
Analysis of LecB and its complex with cinnamide by circular
dichroism (CD) spectroscopy could yield insight on conforma-
tional changes induced by ligand binding. Interestingly howev-
er, we did not observe significant differences in the CD spectra
of LecB with 3 or LecB alone, and both spectra were indicative
of beta-sheets as the predominant secondary structure (Fig-
ure 6A). Then, a thermal denaturation of LecB in presence and
absence of cinnamide 3 was performed to assess the influence
of 3 on protein stability (Figure 6B). The CD spectrum of LecB
was stable between 378C and up to above 908C, and no de-
fined melting point (Tm) could be determined as shown in Fig-
ure 6B for the ellipticity at 229 nm. In presence of com-
pound 3, only slight differences were detected with somewhat
bigger changes without ligand. Further attempts to analyze Tm
of LecB with a set of compounds by thermal shift analysis
using SYPROꢁ Orange also failed to give signals for unfolded
After an extensive search for crystallization conditions of
LecB with cinnamide 3, we finally obtained protein crystals
which diffracted to 1.6 Š and allowed structure determination
(Figure 8, Table 3). Both, the mannose part and the cinnamide
residue were well defined and present in every binding site of
the LecB tetramer (Figure 8A). Interestingly, this crystal struc-
ture shows a surface binding of the cinnamide as opposed to
the computational model suggesting the intercalation of the
aromatic moiety into the protein.[25] The asymmetric unit con-
tains one tetramer of LecB with one ligand molecule per mo-
nomer. In the overlay of ligand binding sites, a high variability
between the ligand conformation in the four binding sites can
be observed (Figure 8A). The mannose residue always adopts
the same binding pose, whereas the cinnamide side chain is
oriented in three different orientations. After closer examina-
tion of the structure, we identified extended p–p stacking con-
tacts between the cinnamide part of two ligands from a neigh-
boring LecB tetramer in the crystal lattice. In only one out of
the four binding sites, the cinnamide substituent is in contact
with the protein surface, forming van der Waals contacts with
Gly97 and Thr98 and a water mediated hydrogen bond with
Ser23 (Figure 8B). In the other three binding sites, the cinnam-
ic acid residues form extended interligand p–p stacking con-
tacts in the crystal lattice. It is well known that crystal lattice
contacts can induce certain conformations of ligands or flexi-
ble regions in proteins, which sometimes do not occur in solu-
tion.[34] In solution, however, the three binding modes lacking
protein contact of the cinnamide are unlikely to occur. The
binding mode observed for the fourth pose with the cinna-
mide having a lipophilic contact with the protein surface could
explain the flat SAR, that is a narrow range of inhibitory activi-
ties, obtained.
In order to further understand the distinctly different charac-
teristics of the interaction of LecB with the cinnamide ligands
observed by protein NMR spectroscopy, we analyzed inhibitor-
mediated aggregation of LecB. Besides intermediate exchange
phenomena or protein unfolding, aggregation also leads to
loss in observed signal intensity by NMR spectroscopy. To
quantify the compounds’ effect on protein aggregation, we
Figure 6. A) CD spectroscopy of LecB (35 mm) in the presence or absence of
compound 3 (100 mm) gave identical CD spectra indicative for beta-sheet
secondary structure. B) thermal unfolding followed by CD spectroscopy at
229 nm was not indicative of a destabilization of the protein in presence of
3.
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tested in absence of LecB were
also translucent across the entire
concentration range. We thus
concluded that unsubstituted
cinnamide 3 dimerizes or even
oligomerizes at high concentra-
tions. These oligomers may act
as multivalent anchor points
that attract individual LecB tet-
ramers and promote aggrega-
tion and precipitation. This ag-
gregation can explain the global
vanishing of signal intensity in
1H,15N-TROSY-HSQC NMR experi-
ments which was observed for 3.
Introduction of the two me-
thoxy-substituents in 7b pre-
vents aggregation of LecB prob-
ably because the methoxy
groups prevent strong p–p-
stacking-mediated oligomeriza-
tion of compound 7b itself.
Conclusions
Cinnamides of d-mannose, such
as 3, were previously identified
as potent glycomimetic inhibi-
tors of LecB, a virulence factor
and important biofilm compo-
nent of the opportunistic patho-
gen P. aeruginosa.[25] Based on
microcalorimetry data and mas-
sive line broadening observed in
protein NMR spectroscopy sig-
nals in the presence of 3, which
may result from global changes
in the protein structure and/or
aggregation of the protein, we
proposed an intercalation bind-
ing model. Here, we report an
extensive SAR study of substitu-
ents at the cinnamide residue of
3 and their influence on binding
affinity to LecB. Although
a rather flat SAR was obtained,
binding affinity of this class of
compounds could be further im-
1
Figure 7. Binding of dimethoxycinnamide 7b to LecB was analyzed by H,15N-TROSY-HSQC NMR experiments in
the absence (blue) and presence (red) of 7b. A) Spectrum of 500 mm 15N-labeled LecB without addition of ligand.
B) Spectrum of 500 mm 15N-labeled LecB in the absence (blue) and in presence (red) of 1 equivalent 7b. Upon ad-
dition of 7b, the intensities of about 15 peaks decreased, and a number of new crosspeaks appeared. This is indi-
cative of chemical shift perturbations of a defined binding that interacts with 7b in the slow-exchange regime.
performed titration experiments with a constant concentration
of LecB and measured aggregation via light scattering at
600 nm (OD600). Surprisingly, we did not observe measurable
light scattering for dimethoxycinnamide 7b between 14 mm
and 1.7 mm, but intense and spontaneous aggregation for cin-
namide 3 at 510 mm and above, which could explain the dis-
tinct differences observed by NMR spectroscopy (Figure 9). Sul-
fonamide 2, as well as the gluco-cinnamide 10, showed no
light scattering under identical conditions; all compounds
proved for LecB. The dimethoxy-substituted compound 7b is
the most potent derivative with a seven-fold higher affinity
than 1. The established SAR at the cinnamide residue was,
however, not sufficient to explain the previously proposed in-
tercalation model of the binding of 3 to LecB. We therefore
further analyzed the binding of 3 to LecB using a number of
biophysical methods.
First, the synthesis and biochemical evaluation of the gluco-
analog 10 of manno-cinnamide 3 showed that the inhibition of
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LecB by compound 3 is specific
and strictly carbohydrate depen-
dent. Furthermore, the data
showed that the inhibitory effect
on LecB does not result from an
unspecific detergent-like effect
of these amphiphilic com-
pounds.
Kinetic analysis by surface
plasmon resonance of the inter-
action of LecB with the two
manno-cinnamides studied here,
3 and 7b, showed comparable
binding kinetics for both com-
pounds. Very slow on rates close
to the detection limit of the SPR
machine, as well as comparable
intermediate off rates were de-
tected for both compounds. This
kinetic data suggests a similar
mode of action for the structur-
ally highly related compounds
Figure 8. A) The crystal structure of the complex of 3 with LecB at 1.6 Š shows a surface binding of the cinnamide
residue to the protein. The superposition of all four binding sites indicates coordination of the cinnamic acid resi-
due to the protein in one monomer and no interaction with LecB in three of the four monomers. The latter results
from extended interligand stacking interactions in neighboring binding sites due to the crystal packing. B) Surface
for monomer 2 with electron density for 3: the cinnamide substituent forms lipophilic interactions with Gly97 and
Thr98 and one water-mediated hydrogen bond via the carbonyl oxygen with Ser23.
cinnamide 3 and dimethoxycinnamide 7b. However, the inter-
1
action study using H,15N-TROSY HSQC NMR spectra of LecB in
Table 3. Crystal structure data of the complex of 3 with LecB.
presence of 7b showed distinct shifts of about 15 resonances
and was therefore in clear contrast to the behavior of its un-
substituted analog 3. After extensive search for crystallization
conditions for the complex of LecB and cinnamide 3, we suc-
ceeded in determining the crystal structure of its complex. Sur-
Data
Beamline (wavelength [Š])
Spacegroup
BM30A/0. 979618
P1
Unit cell dimensions
a, b, c [Š]
44.95, 51.11, 52.35
a, b, g [8]
101.80, 99.40, 115.95
Resolution (outer shell) [Š]
Measured/unique reflections
Average multiplicity
Rmerge
Completeness [%]
Mean I/sI
39.25–1.60 (1.63–1.60)
193815/50076
3.9 (3.7)
0.035 (0.179)
96.6 (90.0)
25.4 (6.1)
CC1/2
0.999 (0.936)
Refinement
Rcryst/Rfree
16.7/20.4
47532/2541
0.0144
1.63
0.102
nb reflections/free reflections
Rmsd bonds [Š]
Rmsd angles [8]
Rmsd chiral [Š3]
Atoms (chain)
Protein
A
851
7.4
175
18.7
23
8.4
2
6.2
B
859
8.3
153
19.8
23
8.3
2
7.0
C
856
7.9
165
21.1
23
9.2
2
6.9
D
838
8.1
128
20.2
23
10.0
2
6.8
Bfac [Š2]
Water molecules
Bfac, [Š2]
Ligand
Bfac, [Š2]
Calcium
Bfac, [Š2]
Ramachandran (Molprobity)
Allowed
Favored
Outliers
100%
97.3%
0%
Figure 9. Aggregation assay of LecB (133 mm) with ligands (14 mm to
1.7 mm). Aggregation at OD600 observed for 3 at the two highest concen-
trations. No aggregation observed for 2, 7b, and 10.
PDB code
5A3O
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solvent peaks as internal standard.[35] Multiplicities were specified
as s (singlet), d (doublet), t (triplet), or m (multiplet). The signals
were assigned with the help of H, H-COSY, and DEPT-135-edited
1H, 13C -HSQC experiments. High-resolution mass spectra (HRMS)
were obtained on a Bruker micrOTOF II ESI spectrometer, and the
data were analyzed using DataAnalysis from Bruker.
prisingly, a surface localization of the cinnamide residue in 3
was detected with defined lipophilic interactions and one di-
rected hydrophilic hydrogen bond with LecB. It was not until
we performed aggregation assays with the compounds stud-
ied and LecB, that we could fully explain the nature behind
the effects observed. Only unsubstituted cinnamide 3 aggre-
gated LecB at high ligand concentrations, similar to those used
in NMR experiments. When two methoxy substituents were in-
troduced into the structurally highly similar compound 7b, ag-
gregation of LecB was no longer observed. Presumably, self-ag-
gregation of compound 3 at high concentrations in aqueous
solution via its lipophilic cinnamide moiety lead to an apparent
multivalency, which precipitated tetravalent LecB. Both cinna-
mides showed comparable very slow on-rates in the surface
plasmon resonance (SPR) experiments with LecB, and therefore
similar binding modes of 3 and 7b are plausible. A possible
conformational change of the protein accounting for these
slow on rates is unlikely due to identical orientations of the
protein in the crystal structures of the apoprotein,[17] LecB with
2[25] or LecB with 3 (Figures S1 and S2 in the Supporting Infor-
mation). The overlay of the crystal structures of LecB and sulfo-
namide 2 with cinnamide 3 also demonstrates the different
areas on the lectin targeted by the sulfonamide and the cinna-
mide residue, respectively (Figure S2 in the Supporting Infor-
mation).
1
1
The NMR and MS data of all synthesized compounds as well as re-
action times and mass yields (Table S1) can be found in the Sup-
porting Information.
General procedure for amide couplings
Methyl 6-amino-6-deoxy-a-d-mannopyranoside[25] (50 mg, max.
0.26 mmol), triethylamine (54 mL, 1.5 equiv), and in case of free
acids, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochlo-
ride (EDC·HCl, 73 mg, 1.5 eq), were dissolved in dry dimethylforma-
mide (DMF, 1.5 mL) and cooled to 08C. Then, the carboxylic acid,
(0.31 mmol, 1.2 equiv) dissolved in DMF (0.5 mL) was added drop-
wise under argon. After stirring for 1 h at 08C, the reaction was al-
lowed to warm to rt and was further stirred for 1–3 days. The reac-
tions were quenched with saturated aqueous NH4Cl (10 mL) and
extracted with EtOAc (1”30 mL, 6”10 mL). The combined organic
layers were dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by MPLC on silica with CH2Cl2/EtOH or
EtOAc/EtOH.
Methyl 6-(2-chlorocinnamido)-6-deoxy-a-d-mannopyranoside (5a)
was obtained from 2-chlorocinnamic acid as a white solid (30%
over two steps).
Based on these experiments and the crystal structure ob-
tained, we could revise the initially proposed intercalation
mode for cinnamide-based LecB inhibitors. The knowledge
gained from this study now allows the structure-based design
of follow-up derivatives of the highly potent cinnamide-based
class of glycomimetics as LecB inhibitors. Importantly, all glyco-
mimetics tested showed extended receptor residence times
with half-lives in the 5–20 min range. In contrast, the rapid ki-
netics of natural carbohydrate ligands with half-lives in the
45 s range hamper their use as therapeutics. Thus, the glycomi-
metics described here provide an excellent basis for future de-
velopment of anti-infectives.
Methyl
6-(2-hydroxycinnamido)-6-deoxy-a-d-mannopyranoside
(5b) was obtained from 2-hydroxycinnamic acid as a white solid
(13% over two steps).
Methyl
6-(2-methoxycinnamido)-6-deoxy-a-d-mannopyranoside
(5c) was obtained from 2-methoxycinnamic acid as a white solid
(56% over two steps).
Methyl 6-(2-methylcinnamido)-6-deoxy-a-d-mannopyranoside (5d)
was obtained from 2-methylcinnamic acid as a white solid (52%
over two steps).
Methyl 6-(2-nitrocinnamido)-6-deoxy-a-d-mannopyranoside (5e)
was obtained from 2-nitrocinnamic acid as a white solid (52% over
two steps).
Experimental Section
Chemical synthesis
Methyl 6-(2-aminocinnamido)-6-deoxy-a-d-mannopyranoside (5 f).
5e (64 mg, 0.174 mmol) was dissolved in degassed H2O, and
FeSO4·7H2O (7 eq) and NH4OH solution (580 mL, 25%) were added
and stirred under argon atmosphere for 30 min. The suspension
was filtered, unsolved residue was washed with NH4OH solution
(5”2 mL, 5%), and solvent was removed in vacuo. After purifica-
tion by MPLC, 5 f was obtained as a yellowish solid (45 mg,
0.133 mmol, 77%).
Thin layer chromatography (TLC) was performed using silica-
gel 60-coated aluminum sheets containing fluorescence indicator
(Merck KGaA, Darmstadt, Germany) using UV light (254 nm) and by
charring either in anisaldehyde solution (1% v/v 4-methoxybenzal-
dehyde, 2% v/v concentrated H2SO4 in EtOH), in aqueous KMnO4
solution or in
a molybdate solution (a 0.02m solution of
Ce(NH4)4(SO4)4·2H2O and (NH4)6Mo7O24·4H2O in aqueous 10%
H2SO4) with heating. Medium pressure liquid chromatography
(MPLC) was performed on a Teledyne Isco Combiflash Rf200
system (Lincoln, USA) using pre-packed silica gel 60 columns from
Teledyne Isco, SiliCycle, or Macherey–Nagel. Commercial chemicals
and solvents were used without further purification. Deuterated
solvents were purchased from Eurisotop (Saarbrücken, Germany).
Nuclear magnetic resonance (NMR) spectroscopy was performed
on a Bruker Avance III 400 UltraShield spectrometer (Bruker Biospin
GmbH, Rheinstetten, Germany) at 400 MHz (1H) or 101 MHz (13C).
Chemical shifts are given in ppm and were calibrated on residual
Methyl 6-(3-chlorocinnamido)-6-deoxy-a-d-mannopyranoside (5g)
was obtained from 3-chlorocinnamic acid as a white solid (54%
over two steps).
Methyl
6-(3-hydroxycinnamido)-6-deoxy-a-d-mannopyranoside
(5h) was obtained from 3-hydroxycinnamic acid as a white solid
(69% over two steps).
Methyl
6-(3-methoxycinnamido)-6-deoxy-a-d-mannopyranoside
(5i) was obtained from 3-methoxycinnamic acid as a white solid
(65% over two steps).
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Methyl 6-(3-methylcinnamido)-6-deoxy-a-d-mannopyranoside (5j)
was obtained from 3-methylcinnamic acid as a white solid (50%
over two steps)
Recombinant expression and purification of P. aeruginosa
LecB
The protein was expressed and purified as described recently[25]
using E. coli BL21 (DE3) and plasmid pET25pa2l.[36] The purified and
lyophilized protein was dissolved in Tris-buffered saline supple-
mented with calcium (TBS/Ca: 20 mm Tris, 137 mm NaCl, 2.6 mm
KCl at pH 7.4 supplemented with 0.1 or 1 mm CaCl2) before use,
and the concentration was determined by UV spectroscopy at
Methyl 6-(3-nitrocinnamido)-6-deoxy-a-d-mannopyranoside (5k)
was obtained from 3-nitrocinnamic acid as a white solid (49% over
two steps)
Methyl 6-(3-aminocinnamido)-6-deoxy-a-d-mannopyranoside (5l).
5k (64 mg, 0.174 mmol) was dissolved in degassed H2O, and
FeSO4·7H2O (7 eq) and NH4OH solution (580 mL, 25%) were added
and stirred under argon atmosphere for 30 min. The suspension
was filtrated, unsolved residue was washed with NH4OH solution
(5”2 mL, 5%), and solvent was removed in vacuo. After purifica-
tion by MPLC, 5l was obtained as yellowish solid (39 mg,
0.143 mmol, 66%).
280 nm using a molar extinction coefficient of 6990mÀ1 cmÀ1 [37] Re-
.
combinant expression and purification of 15N-labelled LecB was
performed using 15NH4Cl as described[25] following a procedure by
Marley et al.[38]
Competitive binding assay
The competitive binding assay based on fluorescence polarization
was performed as previously described by Hauck et al.[25] Briefly,
a stock solution of LecB (20 mL of 225 nm) and fluorescent reporter
ligand N-(fluorescein-5-yl)-N’-(a-l-fucopyranosyl ethylen)-thiocarba-
mide (15 nm) in TBS/Ca were mixed with serial dilutions (10 mL of
1 mm to 12.8 nm) of testing compounds in TBS/Ca in triplicates.
After addition of the reagents, the black 384-well microtiter plates
(Greiner Bio-One, Germany, cat. no. 781900) were incubated for 8–
22 h at rt in a humidity chamber. Fluorescence emission parallel
and perpendicular to the excitation plane was measured on a Pher-
aStar FS (BMG Labtech, Ortenberg, Germany) plate reader or on
a Tecan INFINITE F500 plate reader (Tecan Group Ltd., Männedorf,
Switzerland) with excitation filters at 485 nm and emission filters at
535 nm. For measurements on the Tecan INFINITE F500 plate
reader, the G-factor was set on 0.92154 and the gain to 80. The
measured intensities were decreased by buffer values, and fluores-
cence polarization was calculated. The data were analyzed using
BMG Labtech MARS software (v.3.01R2, 2013) and/or with Graph-
Pad Prism (GraphPad Software, Inc., La Jolla, USA) and fitted ac-
cording to the four-parameter variable-slope model. Bottom and
top plateaus were defined by the standard compounds l-fucose
and methyl a-d-mannoside, respectively, and the data was reana-
lyzed with these values fixed. A minimum of three independent
measurements of triplicates each was performed for every ligand.
Methyl 6-(4-chlorocinnamido)-6-deoxy-a-d-mannopyranoside (5m)
was obtained from 4-chlorocinnamic acid as a white solid (46%
over two steps).
Methyl
6-(4-hydroxycinnamido)-6-deoxy-a-d-mannopyranoside
(5n) was obtained from 4-hydroxycinnamic acid as a white solid
(20% over two steps).
Methyl
6-(4-methoxycinnamido)-6-deoxy-a-d-mannopyranoside
(5o) was obtained from 4-methoxycinnamic acid as a white solid
(55% over two steps).
Methyl 6-(4-methylcinnamido)-6-deoxy-a-d-mannopyranoside (5p)
was obtained from 4-methylcinnamic acid as a white solid (52%
over two steps).
Methyl 6-(4-nitrocinnamido)-6-deoxy-a-d-mannopyranoside (5q)
was obtained from 4-nitrocinnamic acid as a white solid (74% over
two steps).
Methyl 6-(4-aminocinnamido)-6-deoxy-a-d-mannopyranoside (5r).
5q (48 mg, 0.13 mmol) was dissolved in degassed H2O, and
FeSO4·7H2O (7 eq) and NH4OH solution (430 mL, 25%) were added
and stirred under argon atmosphere for 30 min. The suspension
was filtered, unsolved residue was washed with NH4OH solution
(5”2 mL, 5%), and solvent was removed in vacuo. After purifica-
tion by MPLC, 5r was obtained as a yellowish solid (29 mg,
0.087 mmol, 66%).
Microcalorimetry
Isothermal titration calorimetry (ITC) was performed on a MicroCal
ITC200 (GE Healthcare, Freiburg, Germany), and the data was ana-
lyzed using the MicroCal Origin software (v.7.0, 2009). LecB (300–
360 mm) was dissolved in TBS/Ca (the concentration was verified
by UV spectroscopy, see above) and placed in the sample cell at
258C. The titration was performed with a solution of 7b (3.0–
3.5 mm) in the same buffer. After one preinjection (0.4 mL), 19 injec-
tions of 2 mL and 4 s each with a spacing of 460 or 480 s were per-
formed. Three independent titrations were run.
Methyl 6-(2-naphtoylamido)-6-deoxy-a-d-mannopyranoside (6) was
obtained from 2-naphtoic acid as a white solid (41% over two
steps).
Methyl 6-(3,4-methylenedioxycinnamido)-6-deoxy-a-d-mannopyra-
noside (7a) was obtained from 3,4-methylenedioxycinnamic acid in
as a white solid (59% over two steps).
Methyl 6-(3,4-dimethoxycinnamido)-6-deoxy-a-d-mannopyranoside
(7b) was obtained from 3,4-dimethoxycinnamic acid as a white
solid (33% over two steps).
Surface plasmon resonance (SPR) measurements
Methyl 6-(cinnamido)-6-deoxy-a-d-glucopyranoside (10). Methyl 6-
azido-6-deoxy-a-d-glucopyranoside[30] (9, 270 mg, 1.23 mmol) was
dissolved in MeOH (12 mL) and stirred under hydrogen atmos-
phere (1 bar) with 10% Pd/C (30 mg, 10 mol%) at rt for 4 h. The
mixture was filtered through celite, and the solvent was removed
under reduced pressure to give methyl 6-amino-6-deoxy-a-d-glu-
copyranoside[30] (208 mg) as colorless solid that was subsequently
coupled under standard conditions with cinnamic acid (43% over
two steps).
SPR binding experiments were performed on a Biacore X100 in-
strument (GE Healthcare, Freiburg, Germany) at 258C in Hepes-
buffered saline (HBS: 10 mm Hepes/NaOH, pH 7.4, 150 mm NaCl,
0.05% Tween 20) at a flow rate of 30 mL minÀ1. LecB was biotinylat-
ed using EZ-Link NHS-LC-LC-Biotin (Pierce, cat. no. 21343) following
the manufacturer’s conditions. Streptavidin (7000 RU) was immobi-
lized on a research-grade CM5 chip using standard procedures,
and 4200 RU of biotinylated LecB were captured on channel 2.
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Binding experiments were performed with the LecB-captured chan-
nel 2, and plots represent the subtracted data (channel 2–
channel 1). Binding studies consisted of the injection (association
180 s, dissociation 180 s) of various concentrations of inhibitor
(120 nm–75 mm for compounds 2, 3, and 7b, or 3 mm–1.8 mm for
1) in the single cycle mode. Binding was measured as resonance
units over time after blank subtraction, and data were then evalu-
ated by using the Biacore X100 evaluation software (version 2.0).
manually. The stereochemical quality of the models was assessed
with the program Molprobity,[44] and coordinates were deposited
in the Protein Data Bank under code 5A3O.
Aggregation assay
A stock solution of LecB (40 mL, 200 mm in TBS/Ca) was mixed with
serial dilutions of testing compounds (20 mL of final test concentra-
tions of 1.7 mm to 13.5 mm) into a transparent F-bottom 384-well
microtiter plate (Greiner Bio-One, Germany, cat. no. 781901) in du-
plicates. The highest concentration of each inhibitor was included
without LecB in duplicates as control. Absorbance was measured
on a PheraStar FS (BMG Labtech, Germany) plate reader at 600 nm
after 30 min and 4 h of incubation at rt and did not show differen-
ces between the two time points. The data were analyzed using
BMG Labtech MARS software and GraphPad Prism.
Biomolecular NMR spectroscopy
For NMR experiments, 15N-LecB (500 mm) in TBS/Ca was supple-
mented with 5% D2O (Eurisotop, Germany). NMR spectra were
measured in standard NMR tubes (5 mm) in the presence and ab-
sence of ligand (250 or 500 mm) at 300 K. NMR experiments were
performed with
a Bruker Avance III 600 MHz spectrometer
equipped with an inverse H/C/N-TCI-cryoprobe (5 mm) with active-
ly shielded z-gradient. 1H,15N-TROSY-HSQC spectra were acquired
with 256 increments in the indirect dimension with 16 scans per in-
crement. All NMR spectra were processed and analyzed with Bruk-
er’s TopSpin software (version 3.0).
Acknowledgements
The authors are grateful to Holger Bußkamp (University of Kon-
stanz) and Michael Hofmann (Helmholtz Institute for Pharma-
ceutical Research Saarland, HIPS) for HRMS measurements, to Dr.
Sascha Baumann (HIPS) for fruitful discussions, and Emilie Gillon
(Centre de Recherches sur les MacromolØcules VØgØtales,
CERMAV, Grenoble) for support in crystallization experiments.
Crystal data were collected at the European Synchrotron Radia-
tion Facility on beamline FIP-BM30A (proposal 20120846), and
the authors thank David Cobessi for assistance in using the
beamline. The authors also thank the Helmholtz Association
(A. T. , grant no. VH-NG-934), the Konstanz Research School
Chemical Biology (R. S. and A. T.), the Zukunftskolleg (A. T.), and
the Deutsche Forschungsgemeinschaft (A. T. , grant no. Ti756/2-1)
for financial support. A. I. acknowledges support from GDR Pseu-
domonas and Labex ARCANE (ANR-11-LABX-OO3). The funders
had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Circular dichroism (CD) spectroscopy
Protein samples for CD measurements and melting assays were pu-
rified as described above. CD spectra were measured on a JASCO-
J1500 spectropolarimeter (Gross-Umstadt, Germany) equipped with
a PTC-510 Peltier Thermostatted Single Cell Holder using a 1 mm
optical path. Protein samples were prepared as a 34.8 mm solution
in TBS buffer supplemented with 1 mm CaCl2 in a reaction volume
of 300 mL with or without 100 mm compound 3. Scans were per-
formed at 378C over a wavelength range of 200–280 nm (three ac-
cumulations) with a scanning speed of 100 nmminÀ1, 1 nm data
pitch, and 1 nm bandwidth. For thermal denaturation, samples
were prepared as described above. Folded samples were heated
from 378C to 968C with a heating rate of 28C minÀ1. The CD signal
at 229 nm was recorded every 18C.
Crystallization and structure determination
Keywords: carbohydrates · glycoconjugates · glycomimetics ·
LecB/PA-IIL · lectins
Crystals of recombinant LecB from P. aeruginosa were obtained by
the hanging drop vapor diffusion method using 2 mL of drops con-
taining a 50:50 (v/v) mix of protein and reservoir solution at 198C.
The protein at 10 mgmLÀ1 was incubated with 15.3 mm of 3 in
H2O and 100 mm CaCl2 during 1 h at rt prior to co-crystallization.
Clusters were obtained in a solution containing 25% PEG6K, 1m
LiCl, and 0.1m citric acid at pH 3.8. A broken tip was directly
mounted in a cryoloop and flash-frozen in liquid nitrogen. Diffrac-
tion data were collected at 100 K at the European Synchrotron Ra-
diation Facility (Grenoble, France) on beamline BM30 A using
a MARCCD detector. The data were processed using XDS.[39] All fur-
ther computing was performed using the CCP4 suite.[40] Data quali-
ty statistics are summarized in Table 3. The structure was solved by
molecular replacement using PHASER and the tetramer coordinates
of 3ZDV as search model.[25,41] Five percent of the observations
were set aside for cross-validation analysis, and hydrogen atoms
were added in their riding positions and used for geometry and
structure-factor calculations. The structure was refined using re-
strained maximum likelihood refinement in REFMAC 5.8[42] iterated
with manual rebuilding in Coot.[43] Incorporation of the ligand was
performed after inspection of the 2Fo-DFc weighted maps. Water
molecules, introduced automatically using Coot, were inspected
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Received: June 16, 2015
Published online on October 13, 2015
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