STD NMR and molecular modelling insights into interaction of novel mannose-based ligands with DC-SIGN

Article abstract of DOI:10.1039/c5ob01916h

Study of interaction of mannose-based ligands with receptor DC-SIGN using high resolution NMR in combination with molecular modelling showed that four α-d-mannoside ligands interact with the binding site predominantly through the mannose moiety. The other two aromatic groups that are bound to α-d-mannose through a glycerol linker demonstrate interaction that can be related to their substitution pattern. Ligand with naphthyl and meta-substituted phenyl ring exhibited the most favourable binding characteristics. In addition to the predicted hydrophobic interactions of aromatic moieties our results propose new contacts of substituted phenyl moiety in the more polar area of the flat binding site of DC-SIGN and thus offer new possibilities in further designing of novel, more potent DC-SIGN antagonists.

Full text of DOI:10.1039/c5ob01916h

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DOI: 10.1039/C5OB01916H
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STD NMR and molecular modelling insights into interaction of
novel mannose-based ligands with DC-SIGN
Received 00th January 20xx,
Accepted 00th January 20xx
Anita Kotara, Tihomir Tomašičb, Martina Lenarčič Živkovida, Gregor Jugb, Janez Plaveca,c,d,*, and
Marko Anderluhb,*
DOI: 10.1039/x0xx00000x
Study of interaction of mannose-based ligands with receptor DC-SIGN using high resolution NMR in combination with
molecular modelling showed that four α-D-mannoside ligands interact with the binding site predominantly with mannose
moiety. The other two aromatic groups that are bound to α-D-mannose through glycerol linker demonstrate interaction
that can be related to their substitution pattern. Ligand with naphthyl and meta-substituted phenyl ring exhibited the
most favourable binding characteristics. In addition to predicted hydrophobic interactions of aromatic moieties our results
propose new contacts of substituted phenyl moiety in more polar area of flat binding site of DC-SIGN and thus offer new
possibilities in further designing of novel, more potent DC-SIGN antagonists.
www.rsc.org/
endosomes and avoid the host immune system.6, 8, 11, 13, 14 By
inhibiting interaction between DC-SIGN and HIV-1 with DC-
SIGN antagonists pathogen entry could be prevented causing
Introduction
Although significant progress was made in HIV vaccine
strategies and drugs development, HIV infections remain one
of the leading causes of morbidity and mortality worldwide.1-5
Dendritic cells (DCs), hosts professional antigen-presenting
cells that represent first line of innate immune system, play an
important role in HIV-1 infection.6 The pattern recognition
receptors (PRRs) located at the surface of DCs are important
factor in HIV-1 transmission that help subvert DC functions and
escape immune surveillance.4, 7 One of these PRRs that plays
fundamental role in cellular immunity is DC-SIGN (Dendritic
Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-
integrin), a type II transmembrane C-type lectin located
specifically on DCs.8-10 DC-SIGN acts as an adhesion molecule
and binds highly glycosylated proteins present on the surface
of several pathogens, including mannose glycans of the
envelope protein gp120 of HIV-1.11-13 On binding, the complex
of DC-SIGN and HIV-1 is promptly internalized, degraded into
smaller fragments in early endosomes and conjugated with
MHC class-II proteins to initiate a humoral immune response
from T cells, but unfortunately, a small amount of virus
succeeds to escape the normal lytic pathway in DCs’
impediment of the infection of immune defence cells.8, 15-23
Different approaches were applied to design medicinally useful
DC-SIGN antagonists. Even though reported antagonists vary in
structure and properties, they mostly exploit the fact that DC-
SIGN recognises various mannose- and fucose-containing
glycans exposed on the surface of several viruses, bacteria,
yeast and parasites.4, 8, 11, 23 D-mannose (Ki=13.1 mM) and L-
fucose (Ki=6.7 mM) alone bind to DC-SIGN with very weak
affinities, but when incorporated into suitable glycoconjugates
substantially higher affinities can be obtained that offer a
12, 23, 24
promise to prevent the HIV-1 transmission.8,
We
recently reported
mannose-based DC-SIGN antagonists bearing diaryl
substituted 1,3 diaminopropanol or glycerol moieties
incorporated to target the hydrophobic groove of the
receptor.23
mannose entity was presumed to serve as an
a small focused library of branched
-
D
-
anchor for interaction with Ca2+ binding site of the DC-SIGN
carbohydrate recognition domain (CRD). The designed
molecules were evaluated by in vitro assay of the isolated DC-
SIGN extracellular domain for their ability to compete with
HIV-1 gp120 for binding to the DC-SIGN CRD. The two most
potent compounds were found to inhibit the interactions
between gp120 and DC-SIGN with the IC50 values of 40
M and
a.Slovenian NMR center, National Institute of Chemistry, Hajdrihova 19, 1000
50 M, and were among the most potent monovalent DC-SIGN

Ljubljana, Slovenia.
b.Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana,
antagonists reported. Furthermore, the antagonistic effect of
these compounds was evaluated by a one-point in vitro assay
that measures dendritic cell adhesion to a mannan-coated
surface. Again, the same compounds acted as functional
antagonists of DC-SIGN-mediated DC adhesion. The binding
Slovenia
c.
EN-FIST Centre of Excellence, Trg OF 13, 1000 Ljubljana, Slovenia.
d.Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot
113, 1000 Ljubljana, Slovenia.
*Corresponding authors. Fax:+38614258031 E-mail: marko.anderluh@ffa.uni-lj.si
(M. Anderluh), Fax: +38614760300, E-mail: janez.plavec@ki.si (J. Plavec).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
mode of the most potent compound (4) was also studied by
molecular docking and molecular dynamics simulation, which
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Organic & Biomolecular Chemistry
into interactions of our novel ligands with DC-SIGN and
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suggests new possibilities in designDOoI:f10.1n0o3v9e/Cl 5OaBn0t1i9-H16IHV
therapeutics.
Results
Chemistry
DC-SIGN antagonists
while synthesis of
Hydroxybenzoic acid (
1
and
and
5) was first converted to its methyl ester
4
were synthesized as reported,23,
is outlined in Scheme 1. 3-
25
2
3
6
in methanol under acidic conditions. Potassium salt of
compound
6
, which was prepared using potassium hydroxide
to obtain
with 2,3,4,6-tetra-
O-acetyl-α-D-mannopyranosyl trichloroacetimidate23 and with
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as
promoter yielded α-D-mannoside stereospecifically. Since
compound was used as a racemate, compound was
in methanol, was used for epoxide ring opening of
racemic diarylglycerol . Mannosylation of
7
Fig. 1 Chemical structures of monovalent mannose-based ligands.
8
8
pointed that the diarylglycerol part of the molecule likely binds
into the targeted hydrophobic binding site, and provides a
basis for further optimization.23 Unfortunately, the binding site
of DC-SIGN is rather flat and vaguely defined and offers limited
(other than van der Waals) ligand-receptor contacts.
9
8
9
obtained as a mixture of two diastereomers that could not be
separated using flash column chromatography. Deprotection
In the present study we investigated the interactions between
of compound
which was further hydrolysed under alkaline conditions to
yield the final compound
9 using Zemplén method gave final compound 3,
four monovalent mannose
extracellular domain of DC-SIGN (hereinafter referred as DC-
SIGN). Studied ligands 1-4 consist of α mannose connected
-based ligands 1-4 (Fig. 1) and
2
.
-D-
by glycerol linker to different aryl moieties, which are expected
to tune interactions of ligands in hydrophobic groove of the
receptor. The combination of saturation transfer difference
(STD) NMR spectroscopy and molecular modelling has been
used to examine binding propensity of monovalent
glycoconjugates for DC-SIGN at the atomic level of resolution.
STD NMR experiments and epitope mapping
The STD NMR experiments were performed to explore the
binding nature and propensity of ligands 1-4 to interact with
DC-SIGN. Reference and STD spectra exhibited narrow and
well resolved lines (Fig. 2). The significant STD effects observed
for ligands 1-4 unequivocally demonstrated their binding to
the DC-SIGN. In order to increase accuracy of epitope mapping
we used only well resolved and isolated NMR resonances of
the individual ligand, avoiding uncertain contributions in the
case of signal overlap. Quantitative analyses of the STD spectra
were done at receptor-to-ligand ratio of 1:50. Rather high ratio
was used to reduce the effect of cross-rebinding, where a
previously saturated ligand re-enters a binding pocket.
As expected, ligands 1-4 do not exhibit clear differences in
chair conformation of D-mannose sugar moiety regardless of
variation of groups attached to its anomeric carbon. On the
other hand, differences in ligands’ binding affinities can be
correlated with the position of carboxylic group on the phenyl
moiety in 1-3 as well as lipophilicity of each of the ligands.
Binding mode of ligands was additionally evaluated by
molecular dynamic simulations. A good agreement between
NMR and molecular modelling results provides new insights
Scheme 1 Reagents and conditions: a) SOCl2, MeOH, r.t., 24 h; b) (i) KOH, MeOH, r.t., 20 min; (ii) tetrabutylammonium bromide, toluene/N,N-dimethylformamide, 90 °C, 20 h; c)
2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl trichloroacetimidate, TMSOTf, CH2Cl2, 0 °C, then r.t., 24 h; d) NaOMe, MeOH, 1 h, then Amberlite® IR120 H, 10 min; e) 1 M NaOH, EtOH,
r.t., 24 h.
2 | Org. Biomol. Chem., 2012, 00, 1-3
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Fig. 2 1H and STD NMR spectra of 1-4 in the presence of DC-SIGN. NMR spectra were recorded at 25 °C, 2.0 s saturation time, 400 μM concentration of ligand and 1:50 receptor-to-
ligand ratio in 25 mM Tris-d11 (pD=8.0), 150 mM NaCl, 4 mM CaCl2 in 2H2O and 25 % DMSO-d6 at 600 MHz. Resolved signals are labelled according to atom numbering shown in Fig.
3.
For 1-4 the most intense STD signals were observed between δ The aromatic groups exhibited weaker STD effects in
3.3 and 3.9 ppm, which suggested that mannose moiety was in comparison to mannose moiety regardless of variations in
the closest contact with the surface of DC-SIGN. However, attached aryl entities. Both phenyl Hg protons of
1 together
quantitative interpretation of STD effects of sugar protons was showed STD effect of 51%. However, in STD spectra one
hampered due to spectral overlap with the exception of cannot distinguish between individual contributions of two Hg
anomeric Ha proton. Ha proton exhibited well
-resolved signals atoms due to their symmetric arrangement at meta-position of
for all four ligands 1-4. Moreover, it showed the highest phenyl group. Hb, Hc and Hd protons of naphthyl group of
1
relative STD effect among protons that were analysed in exhibited STD effects of 61%, 43% and 53%, respectively.
epitope mapping. Consequently, the signal of Ha proton was Obvious STD effects were seen for other aromatic protons as
set as a reference with the 100% STD effect. Intensities of well.
other signals were expressed relative to the Ha signal (Fig. 3).
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Fig. 3 Epitope mapping for 1-4. The relative degrees of saturation of marked Ha-Hg protons expressed in % are mapped onto the structure and normalized with respect to Ha
proton. STD effect of Ha was arbitrarily set to the 100% in the case of all four ligands. Arrows are used to report the overall STD effects for symmetric groups.
Unfortunately their quantitative elucidation was prevented STD build-up studies
due to spectral overlap. In
2 the phenyl Hf proton showed STD
To precisely map ligand epitopes in close contact with the
receptor we acquired STD build-up curves for 1-4 by collecting
STD spectra at twelve different saturation times ranging from
0.1 to 5.0 s at receptor-to-ligand ratio of 1:50. The maximal
effect of 68% and Hb, Hc and He protons from naphthyl moiety
exhibited the STD effects of 78%, 54% and 50%, respectively.
However, direct comparison of ligands has to be done with
certain degree of caution because of monitoring of protons on
different positions on aromatic groups of ligands, namely Hd in
STD intensities, STDmax, and the saturation rate constants, ksat
were obtained by fitting experimental data to Eq. 2.
Experimental data as well as best fit curves for are shown in
,
1
and He in 2-4 on naphthyl ring, and Hg in
phenyl moiety. In Hb and Hc protons from naphthyl and Hf
from phenyl group showed similar STD effects ranging from
73% to 77%. He proton in exhibited STD effects of 65%.
Experimentally observed STD effects for Hb and Hc protons on
both naphthyl rings in were 90% and 91%, respectively. STD
effects of He protons were markedly weaker (62%). However,
one should keep in mind that Hb, Hc and He protons in could
1 and Hf in 2-4 on
-
1
3
Fig. 4, whereas the corresponding figures for 2-4 can be found
in the Supplementary information (Figs. S2-S4). The agreement
between the experimental data and the fitted model was good
with typically χ2 values below 0.1. A clear build-up of the STD
effects with increasing saturation time can be observed,
reflecting the amplification of the STD (Figs. 4, S4-S6).
At longer saturation times flattening of the build-up curves can
be observed, because of the relaxation rate of saturated ligand
in the free state. For 1 the most prominent STDmax belonged to
3
4
4
not be resolved due to symmetry of the ligand. Observed STD
effects therefore represented the overall contribution to the
binding for both protons.
the Ha proton (4.46), while the corresponding values for
aromatic Hb, Hc, Hd and Hg protons were markedly lower (2.50,
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saturation time (STD2s) from epitope mapping. For ease of
comparison, the values were normalized with respect to Ha as
a 100 (Table 1).
The STD0 values were lower than STD2s effects. The two
exceptions were Hc and Hd protons of
markedly higher STD0 values. STD2s and STD0 values for Hg of
were comparable. The same observations were noticed also
for Hc and He protons of ligand . On the contrary, Hb and Hf
protons of showed significant differences in STD effects. For
ligand , identical STD2s and STD0 values were observed for He
1, which exhibited
1
2
2
3
proton. On the other hand, significantly lower STD0 effects
were detected for Hb, Hc and Hf protons in comparison to STD2s
values. Surprisingly, the lowest STD0 values and the greatest
variances among STD effects were observed for protons of 4.
Characterisation and comparison of binding conformations of 1-4
Fig. 4 STD-amplification factor of 1 as a function of saturation time. Experimental data
The normalized build-up curves of STD effects for individual
STDmax=2.50 ± 0.07, protons were compared to characterise differences in binding
were fitted to
a
rising exponential Eq. 2, to obtain the STDmax and ksat
ksat=1.1 ± 0.1 s-1, R2=0.973;
Hb:
.
Ha: STDmax=4.46 ± 0.1,
ksat=0.96 ± 0.08 s-1, R2=0.983, Hc: STDmax=2.06 ± 0.07, ksat=1.5 ± 0.2 s-1, R2=0.961; Hd:
STDmax=2.49 ± 0.06, ksat=1.5 ± 0.2 s-1, R2=0.982; Hg: STDmax=2.66 ± 0.08, ksat=1.1 ± 0.1 s-1,
R2=0.978). Symbols represent data, whereas solids lines are the mathematical least-
square fits.
modes among ligands 1-4 (Fig. 5). STD effects of analysed
protons within individual ligand at different saturation times
were normalized to STD effect of Ha proton at saturation time
of 5.0 s.
Good correspondence in overall trend between STD build-up
curves was observed for Ha proton in 1-4 (Fig. 5a). One can
observe slight deviation of data points among the ligands at
saturation times between 1.0 and 2.0 s (Fig. 5a). Similar build-
up curves suggest comparable binding conformation of
mannose moiety in 1-4 in the binding site of DC-SIGN.
2.06, 2.49 and 2.66, respectively). Accordingly, Ha proton
should be the one most directly involved in binding and closer
to the receptor, followed by aromatic protons that are
approximately equidistant from the protein surface. Different
ksat values spanning from 0.96 to 1.54 s-1 were calculated for
protons of
1. In the case of 2 the STDmax values of Ha, Hb, Hc, He,
Table 1 Normalized STD intensitiesa
Hf protons were 3.71, 1.30, 1.87, 1.48 and 1.61, respectively. It
is interesting that ksat values among protons of
remarkably and were all around 1.30 s-1. For
2
3
did not vary
the highest
1
2
3
4
Hb
Hc
Hd
He
Hf
Hg
Hb
Hc
Hd
He
Hf
61
43
53
ndb
nd
51
49
62
73
nd
nd
46
78
54
nd
50
68
nd
46
48
nd
45
49
nd
73
75
nd
65
77
nd
59
53
nd
65
58
nd
90
91
nd
62
STDmax of 3.44 was obtained for Ha proton. Significantly lower
values were acquired for aromatic protons, Hb (2.01), Hc (1.58),
He (1.08) and Hf (1.77). Great differences in ksat from 1.19 to
STD2s
2.45 s-1 were determined among protons of
3
. Similarly,
exhibited ksat values ranging between 1.59 and
2.44 s-1. The STDmax values of
were 3.41 for Ha proton and
c
/
protons of
4
/
4
42
38
nd
45
/
1.53, 1.69 and 1.30 for naphthyl Hb, Hc and He protons,
respectively. Once again, results inferred that the Ha proton
was in close contact with the protein and that the mannose
moiety contributed primarily to the binding process.
Furthermore, initial build-up rates were analysed as well.
Initial slopes of the curves at zero saturation time were
derived by multiplication of STDmax and ksat and correspond to
the STD effects (STD0) in the absence of influence of T1
longitudinal relaxation time bias and rebinding effects.We
calculated STD0 for Ha, Hc, Hd, He, Hf and Hg protons and
compared these results with the STD intensities at 2.0 s
STD0
Hg
/
aNormalized intensities for Hb-Hg protons at 2.0 s saturation time with respect to
STD of Ha which was arbitrarily given a value of 100.
bNot determined because of peak overlap.
cLigand does not contain this proton.
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Fig. 5 Overlaid normalized STD build-up curves of individual protons in 1-4. The normalized STD effects of ligand protons are shown as a function of saturation time of a) Ha, b) Hb,
c) Hc, d) Hd/e and e) Hf/g
.
Normalized STD-AF values for Ha proton were the most this proton within individual ligand.
pronounced in all four cases of interaction analysis of 1-4 with comparable outline of build-up curves and reach almost
DC-SIGN. These results suggest that the Ha proton and identical plateau value. Build-up curve of grew in the same
associated mannose sugar serve as a binding initiator and are way, but reached the plateau at a lower value. experienced
crucial for interaction. Comparison of normalized build-up different build-up mode of Hb protons with steeper initial slope
curves for Hb proton indicated different rate of saturation of that flattens at value comparable to Hb proton of
1 and 3 exhibited
2
4
2
.
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Differences in saturation trend originate from contribution of
both Hb protons of , which obviously get saturated in shorter
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DOI: 10.1039/C5OB01916H
4
time but do not contribute predominantly to the binding. The
normalized STD-AF values for Hc proton showed excellent
correspondence with deviation of individual experiments data
points within experimental error (Fig. 5c). These observations
confirmed analogous binding mode of Hc proton of 1-4. Hc
protons of
interaction with the receptor. Both Hc protons of
showed similar plateau value as . However, one should
1
-
3
participate approximately equally in the
4
together
1-3
keep in mind that the contribution of individual Hc proton
would be markedly reduced, suggesting lower binding
propensity of ligand
observed analogous slope outline for Hc protons of
faster rate of saturation in comparison to . The major
differences in build-up curves were observed for Hd/e and Hf/g
protons. The discrepancy for Hd and Hg protons of was
4
. As in the case of Hb protons of
4 we
4
indicating
1-3
1
Fig. 6 Normalized STD-AF curves as a function of ligand concentration in the range from
80 to 800 μM. Experimental data were collected at 25 °C, 2.0 s saturation time on a 600
MHz NMR spectrometer with 8 μM DC-SIGN concentration in 25 mM Tris-d11 (pD = 8.0),
150 mM NaCl, 4 mM CaCl2 and 25% DMSO-d6 in 2H2O. Experimental data were fitted to
expected due to their different position on aromatic rings in
comparison to Hf and He protons of 2-4. On the other hand,
significant alterations in STD effects of He proton of
2 and 3
the Eq.
3
(1: KD=1.6 ± 0.5 mM, R2=0.992, 2: KD=0.96 ± 0.08 mM, R2=0.999, 3:
imply different binding properties of naphthyl group and
highlight sensitivity of binding characteristics of ligands.
KD=0.45 ± 0.05 mM, R2=0.997, 4: KD=4.9 ± 0.7 mM, R2=0.999).
Although, He protons of
build-up curves, direct comparison can be misleading since
curve of represents saturation of two He protons (Fig. 5d).
When comparing structural setup and position of Hd proton of
and He of 2-4 it is evident that Hd proton was closer to the
surface of receptor with respect to He. Furthermore, the
comparison of curves of Hf and Hg protons of phenyl ring from
1-3 showed similar STD build-up (Fig. 5e). Because of the
2 and 4 exhibited comparable STD
Consistently, these results suggest that exchange kinetics was
the fastest for
binding site of DC-SIGN for longer period of time in
comparison to other ligands. The binding affinity of and
was in lower milimolar range with KD values of 0.45 mM and
0.96 mM, respectively. For and weaker binding with KD
4 and the slowest for 3. Thus 3 stayed in the
4
3
2
1
1
4
values of 1.6 mM and 4.9 mM was observed, respectively.
The direct comparison of KD values is difficult as many
experimental factors, such as saturation time, ligand residence
time in the complex, intensity of the signal and accumulation
of saturation in the free ligand affect determination of
receptor-ligand binding affinities by STD NMR spectroscopy.
Therefore, we used binding isotherm of STD-AF initial growth
approach that allows KD evaluation without aforementioned
spurious factors. To verify the sensitivity of the system of
individual ligand and DC-SIGN to saturation transfer, we
analysed STD-AF of 2-4 at different ligand concentrations from
80 to 800 μM at saturation times between 1.0 and 4.0 s and
obtain STD build-up curves The STD0 values as a function of the
ligand concentration were fitted to Eq. 3 to determine KD
values without dependence on the spectral properties of the
observed signals (Fig. S8). Binding isotherm protocol gave
similar KD values in comparison to the titration experiments at
inseparability of two Hg protons of
contribution to STD values we believe that the Hf of
still spatially closer to the hydrophobic groove of DC-SIGN in
comparison to Hg of . Comparative analysis of and
additionally revealed that Hf proton from lies closer to the
receptor with respect to Hf of . Higher lipophilicity of
evidently enables stronger interactions with the receptor. STD
build-up curves comparison for led us to conclusion that
1
and their joint
2
and are
3
1
2
3
3
2
3
1-4
variations in chemical structures of ligands influence their
bound conformations as well as binding modes in the binding
site of DC-SIGN.
Evaluation of binding affinities
Amplification of STD effects with increasing concentration of 1-
4
was used to determine the binding affinities. Receptor-ligand
dissociation constants (KD) were determined with the use of
single-ligand titration experiments that were performed at five
different ligand concentrations from 80 to 800 μM at constant
saturation time of 2.0 s. By monitoring the growth of the STD-
AF upon titration KD values were calculated with the use of the
Eq. 3 (Fig. 6). Experimental data showed that the maximum
STD-AF was not reached for any of ligands even at 800 μM
ligand concentration. This observation is in agreement with
fast exchange of ligands from the bound to the free state. The
variation in exchange kinetics among the ligands is reflected in
different slopes of hyperbolic curves (Fig. 6).
a single saturation time. KD values were 0.90 mM for
2, 0.51
mM for and 3.0 mM for . These KD values confirmed that 3
3
4
exhibits the lowest koff rate and thus spend the longest time as
part of a complex with DC-SIGN.
Molecular docking
Plausible binding modes of compounds (
-3 and ( -3 were first studied by molecular docking
calculations using our previously reported and validated FlexX
protocol.23 The docking binding mode of compound
was
S)-1, (R)-1, (S)-2, (R)-2,
(S
)
R)
4
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Organic & Biomolecular Chemistry
already described.23 DC-SIGN CRD in complex with Man4 (PDB
entry: 1SL4) was selected as receptor for ligand docking under
defined pharmacophore type constraints. In detail, to place
the core D-mannose residue correctly, the side chain
carboxylate groups of Glu354 and Glu347 were defined as
hydrogen bond acceptors, which interact with 3- and 4-
hydroxyl groups of the core D-mannose in the crystal
View Article Online
DOI: 10.1039/C5OB01916H
structure. Additionally,
a pharmacophore with spherical
coordination around Ca2+ ion was defined to correctly consider
the complex interactions between Ca2+ ion and 3- and 4-
hydroxyl group of D-mannose. The D-mannose residue of the
studied antagonists 1-4 is predicted to bind to the DC-SIGN
Ca2+-binding site. In the case of (
S)-3, (R)-3 (Fig. 7) and 4 the
a)
binding of the D-mannose residue is in agreement with the
defined pharmacophore type constraints, whereas in the case
of benzoic acid derivatives (S)-1, (R)-1, (S)-2 and (R)-2, the D-
mannose residue is rotated in a way that only 2-hydroxyl
group is in contact with the Ca2+ ion. Such a D-mannose
binding mode does not allow for octacoordination of the Ca2+
ion in the binding site and has, to the best of our knowledge,
not been observed in the crystal structures of DC-SIGN CRD-
ligand complexes. The FlexX-calculated binding modes of both
diastereoisomers of compound 3 show the same binding mode
of the D-mannose residue, which interacts with Ca2+ ion and
forms hydrogen bonds with Glu347, Asn349, Glu354, Asn365
and Lys368 side chains (Fig. 7). The main difference is in the
b)
orientation of the diarylglycerol moiety. In the case of (R)-3
Fig. 7. FlexX docking poses of compounds a) (R)-3 (in yellow sticks) and b) (S)-3 (in cyan
sticks) in DC-SIGN CRD Ca2+-binding site. Protein (PDB entry: 1SL4) is presented in grey
cartoon and Ca2+ ion as a green sphere. For clarity only side chains of amino acid
residues predicted to interact with the docked ligands are shown as grey sticks.
phenyl ring makes hydrophobic contacts with Phe313 and
forms a hydrogen bond between ester carbonyl group and
Ser360 side chain hydroxyl group, whereas the naphthalene
ring points towards the solvent (Fig. 7a). The opposite can be Hydrogen bonds are shown as black dashed lines. Figure was prepared by PyMOL.26
observed in the binding mode of (S)-3, where the naphthalene
ring contacts the Phe313 side chain, while the ester carbonyl
group forms hydrogen bond with the Asn344 side chain (Fig.
7b). In contrast to (R)-3 and (S)-3 both naphthalene rings of
Carboxylic acid moiety of these ligands points toward the
solvent and is not in contact with the protein.
compound
4 are predicted to form hydrophobic and/or π-π
Molecular dynamics simulations
interactions with the side chain of Phe313.23
Recently, we have shown that DC-SIGN CRD is a challenging
target for docking, because of its flat binding site, and careful
selection of the docking software and parameters is needed to
correctly predict the binding of DC-SIGN antagonists.27
Considering the flat DC-SIGN binding site, the use of semi-
flexible docking protocols, where the protein is treated as rigid
and the ligand as flexible, is not sufficient to represent the
dynamic nature of DC-SIGN-ligand interactions in solution.
Moreover, superposition of crystal structures of DC-SIGN CRD
in complex with different ligands shows that some binding site
residues, including Phe313, which is targeted by the
hydrophobic substituents of 1-4, are rather flexible.
In order to evaluate whether other docking tools can correctly
predict the D-mannose binding pose of compounds
1 and 2,
the docking of compounds 1-4 to DC-SIGN CRD was repeated
with our recently reported docking protocol27 using FRED
3.0.1,28-30 as available in OEDocking software (Release 3.0.1,
OpenEye Scientific Software, Inc., Santa Fe, NM, USA;
www.eyesopen.com/). In this case, the Glu347 and Glu354
side chains were defined as hydrogen bond acceptors and the
Asn365 side chain as a hydrogen bond donor to constrain the
placement of the D-mannose moiety. FRED docking poses for
compounds (R)-3 and (S)-3 were found to be very similar to
those obtained by FlexX, positioning the naphthalene and
phenyl rings to the same binding pockets of the binding site
(Figure S9). In contrast to FlexX, FRED was able to correctly
Therefore, to get a better understanding of the binding of
3 to
DC-SIGN CRD, molecular dynamics simulations of DC-SIGN CRD
in complex with (R)-3 or (S)-3 in water were performed.
Considering similar docking poses obtained by FRED and FlexX,
the highest ranked FlexX docking pose for each ligand in
complex with DC-SIGN CRD, as shown in Fig. 7, was selected to
be used as an input for molecular dynamics simulation. The
complexes were first solvated using TIP3P water model and
place the D-mannose residue of (S)-1, (R)-1, (S)-2 and (R)-2,
allowing the octacoordination of Ca2+ by interactions with D-
mannose 3- and 4-hydroxyl groups (Fig. S10). Both aromatic
moieties of (
contacts and/or π-π interactions with Phe313 side chain (Fig.
S10), similarly as observed in the case of compound 4 23
S)-1, (R)-1, (S)-2 and (R)-2 form hydrophobic
.
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then subjected to molecular dynamics simulation, which linker slightly rotated and oriented the phenyl rinVgieowfAtrthicele O(Sn)li-n3e
DOI: 10.1039/C5OB01916H
consisted of three consecutive steps: (i) solvent equilibration toward Arg345, while the naphthyl ring remained in contact
for 0.5 ns with ligand and protein constrained harmonically with Phe313 side chain (Fig. 9c), as observed in the docking
around the initial structure; (ii) equilibration of the complete conformation (Fig. 7b). During the next few nanoseconds of
system for 1 ns with ligand and protein released; (iii) an the simulation, the naphthyl ring of (S)-3 started moving away
unconstrained 10 ns production run to allow the protein and from the Phe313 (Fig. 9d) and finally reached Asn344/Arg345
the ligand to position themselves according to physical forces site (Fig. 9e). Such a rotation of the glycerol linker resulted also
between them. Physical stability of the simulations was in reorientation of the phenyl ring, which left the
monitored by temperature, volume and total energy vs time Asn344/Arg345 site and moved toward Phe313 (Fig. 9d, e);
plots. All these parameters of the system were stable during however, it was mainly in contact with solvent.
the production run of the simulation. Structural stability of the To evaluate whether the observed differences in (
system during the simulation was monitored by measuring the behaviour during the first MD simulation are relevant, the
RMSD values of protein backbone atoms during the simulation MD was repeated twice more for each DC-SIGN-ligand
R)-3 and (S)-
3
(Fig. 8a and 9a).
complex. In all cases protein backbone was structurally stable,
Interestingly, R and S isomers of
3
in complex with DC-SIGN reflected by the low RMSD values (Fig. 8a and 9a). Ligand
showed significantly different behaviour during the molecular RMSD values and visual inspection of the trajectory during
dynamics simulations. After 0.3 ns of the production run D- different MD runs of DC-SIGN-(S)-3 complex showed that the
mannose 4-hydroxyl group of (R)-3 slowly began to lose D-mannose moiety is tightly bound to Ca2+ and neighbouring
contact with the Ca2+ ion (Fig. 8c). For the next 0.5 ns only D- amino acid residues, while the diaryl substituted glycerol linker
mannose 3-hydroxyl group of (R)-3 was in contact with Ca2+ finally adopts conformation similar to that presented in Fig. 9e.
ion (Fig. 8d). Since Ca2+ ion was not octacoordinated anymore, In contrast to the first MD simulation run of DC-SIGN-(
R)-3
the complex became unstable and the ligand left the binding complex, the second and third runs showed its better stability.
site (Fig. 8e), which is also reflected by the large RMSD values Towards the end of the second production run the D-mannose
of the ligand (Fig. 8b). In contrast, molecular dynamics moiety of (R)-3
began to lose contact with Ca2+, which might
simulation showed greater stability of the DC-SIGN CRD-(S)-3 eventually lead to dissociation of the complex, as presented in
complex (Fig. 9). Although quite high RMSD values for the Fig. 8d,e. In the third run, the complex remained fairly stable,
ligand were observed (Fig. 9b), D-mannose moiety of (S)-3 reflected by the relatively low RMSD values (Fig. 8b). D-
remained tightly bound to Ca2+ ion and other interacting Mannose residue of ( was constantly in contact with Ca2+,
R)-3
residues, as described above. Glycerol linker with the two while the diarylglycerol moiety showed conformational
aromatic moieties showed much greater flexibility than D- flexibility.
mannose. At the beginning of the production run, glycerol
a)
b)
c)
d)
e)
Fig. 8. a) Backbone RMSD and b) ligand RMSD vs time plots for molecular dynamics simulations of DC-SIGN CRD in complex with (R)-3. Molecular dynamics snapshot (run 1) of (R)-
3 in complex with DC-SIGN CRD at c) 0.3 ns, d) 0.8 ns and e) 4.5 ns of the production run.
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DOI: 10.1039/C5OB01916H
Organic & Biomolecular Chemistry
ARTICLE
a)
b)
c)
d)
e)
Fig. 9. a) Backbone RMSD and b) ligand RMSD vs time plots for molecular dynamics simulations of DC-SIGN CRD in complex with (S)-3. Molecular dynamics snapshot (run 1) of (S)-3
in complex with DC-SIGN CRD at c) the beginning d) after 1.3 ns and e) 7.5 ns of the production run.
The final pose of production run was very similar to that of (
3
S)- results correlate well with the known binding specificity of DC-
presented in Fig. 9d, with naphthalene ring pointing toward SIGN for D-mannose sugar.23, 24, 31
solvent and the phenyl ring interacting with Arg345 side chain. In accord with group epitope mapping results, STD build-up
studies revealed the tightest involvement of Ha (and thus
mannose group) in DC-SIGN interaction. Normalized build-up
curves for Ha-Hg protons of 1-4 exposed structure
Discussion
modification-related differences in binding modes. These
curves were consistent in the case of Ha proton, indicating that
binding configuration of mannose sugar was very similar for all
studied ligands. The latter was confirmed for Hc proton on
naphthyl ring as well. Normalized STD build-up curves with
great agreement confirmed analogous binding mode of Hc
proton regardless of structure variations of ligands. On the
other hand, build-up curves of Hb proton indicated altered
involvement of this proton in ligand-receptor interaction.
Results suggest that Hb proton is in closer contact with
Present study delivers new structural and mechanistic details
at the atomic level of binding interactions of novel monovalent
antagonists with DC-SIGN. Acquired STD NMR data on 1-4
unequivocally demonstrated their interactions in the binding
site of the receptor. The STD group epitope mapping revealed
that mannose group formed the strongest interactions with
the DC-SIGN and was in the closest contact to the receptor in
the case of ligands 1-4 (Fig. 3). Individual STD contributions
and interaction involvements of sugar protons were
inseparable due to spectral overlap of signals between δ 3.3
and 3.9 ppm. On the other hand, anomeric Ha proton of 1-4
receptor in
from comparison of Hd/e protons it is obvious that Hd of
closer to the surface of the receptor with respect to He of
One should keep in mind that in the case of all naphthyl
protons in individual contributions would be markedly
1
and
3
in comparison to
2
and
4
. Additionally,
1
was
displayed well-resolved signals with δ between 5.0 and 5.2
2-4.
ppm. Additionally, most effective STD transfer to Ha proton of
1-4 was observed among all investigated protons (i.e. Ha-Hg).
Consequently, Ha proton was considered as adequate
representative of strong mannose group interaction. These
4
reduced because of overall symmetry-related STD effects of
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Hb, Hc and He protons. Although, build-up curves of Hb and Hc moiety, which is in most cases the most tightly bound part of
View Article Online
of
4
indicating faster rate of saturation in comparison to
1
-
3
,
the DC-SIGN antagonist. Because of thDeOrIa: 1t0h.e10r39fl/aCt5ODBC0-1S9I1G6NH
maximum STD-AF were reached at lower values. Rapid binding site, there are only few residues that could be targeted
saturation at low STD-AF plateau values is an indicator of fast with the ligand to increase its affinity. In our case, aromatic
exchange kinetics, which is in agreement with exchange moieties for interaction with the Phe313 side chain were
properties of
KD value among
binding affinity of
4
. The latter was also confirmed with the highest introduced to improve the binding by possible
which supports our observations of lowest stacking/hydrophobic interactions, while carboxylates were
introduced to explore possible formation of additional ionic
-
1-4
4
.
While two naphthyl constituents demonstrated low interaction interaction with Arg345 (see Fig. 10). While the docking
tendency, substitution one of naphthyl ring with phenyl calculations predicted the targeted hydrophobic interaction
moiety resulted in moderate improvement of binding between the ligand and DC-SIGN (Fig. 7), molecular dynamics
propensity. Our results indicate that different position of simulations showed that these interactions are probably quite
carboxylic group on phenyl ring influence the binding weak given the observed flexibility of the ligands and low
conformation not only of modified part of ligand but also of stability of the complexes during the molecular dynamics
intact naphthyl ring. When comparing 1 and 2 it is obvious that simulations (Fig. 8 and 9).
the position of carboxylic group on phenyl moiety affect the Therefore, taken together the results of STD NMR and
binding propensity of the ligand. Although, STD-build up curves molecular dynamics simulations, we can conclude that our
show that Hb and Hc protons of
comparison to , the opposite was observed for Hg of
of . Contribution of individual Hg proton of is believed to be combined results obtained by NMR and MD simulations (Fig.
lower than of Hf of . Para-position of carboxylic group on 10). Interaction of 3- and 4-hydroxyl group of D-mannose with
phenyl ring in
displays better binding properties of adjacent DC-SIGN CRD Ca2+-binding site is driven by four key bonds,
naphthyl moiety in comparison to
. However, shift of namely two coordination bonds with Ca2+ ion and two H-bonds
1
lay closer to the receptor in ligands bind rather tightly through D-mannose residue. We
2
1
and Hf proposed a binding model of DC-SIGN- complex based on
3
2
1
2
1
2
carboxylic group from para- to meta- position improved with side chains of Glu347 and Glu354. Additionally, H-bonds
binding propensity of phenyl constituent confirmed by STD between other D-mannose hydroxyl groups and Asn349,
group epitope mapping and evaluation of KD values. Lower KD Asn365 and Lys368 side chains of DC-SIGN were predicted as
value determined for 2 (0.96 mM) in comparison to 1 (1.6 mM) well. Strongest interaction of D-mannose was confirmed by
indicates better binding tendency of overall ligand. First, these highest STD effects for Ha proton, marked with red (Fig. 10).
results imply that, between both constituents, phenyl moiety Other parts of the ligands show moderate flexibility, especially
primarily contributes to binding affinity and plays more glycerol linker that enables rather free conformations and
important role in complex formation. And second, carboxylic binding possibilities of attached aryl moieties. However,
group at meta-position on phenyl ring prolongs the half-life of naphthyl ring was detected in the spatial proximity to Phe313
ligand-receptor complex.
where it forms hydrophobic interactions as predicted.
Furthermore, esterification of carboxylic group at meta-
position on phenyl moiety additionally decreased KD value.
Notable differences in binding properties among
2 and 3 were
observed not only for Hf proton but also for the whole set of
protons on naphthyl ring. Hf proton was after carboxylic group
esterification closer to the surface of the receptor, since STD
effect increased from 68% in
2 to 77% in 3. Greater spatial
proximity was additionally confirmed with higher normalized
STD-AF values for Hf of
3
in regard to
2
. While Hc protons of
2
3
and demonstrated analogous binding involvements, Hb of
3
was shown to be closer to the receptor surface. On the other
hand, in the case of He proton of we observed less intense
contact to the receptor in comparison to . It is evident that
esterification of carboxylic group enhanced the binding
features of which reflected in lowest KD value among
(0.45 mM). Additionally, we confirmed that esterification
3
2
3
1-4
affected the position of the naphthyl ring in the binding site of Fig. 10 Binding model of DC-SIGN-3 complex predicted based on measured STD effects
and molecular modelling results. Surface of DC-SIGN CRD Ca2+-binding site is shown in
DC-SIGN as well.
Our molecular docking and molecular dynamics simulations
showed that structure-based design of novel DC-SIGN
the back (PDB code 1SL424). Ca2+ ion is shown as bigger green sphere, while observed
ligand protons, namely Ha, Hb, Hc, He and Hf, are shown as smaller spheres. Ligand
protons are coloured based on ligand-receptor interproton distance, red displaying the
antagonists based only on docking calculations remains a great
closest and blue presenting the most distant spatial proximity. Hydrogen and
challenge and provides very limited information about the coordination bonds are marked with dashed lines.
protein-ligand interactions in solution. Docking protocol is
usually able to correctly predict the binding of the D-mannose
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ARTICLE
Organic & Biomolecular Chemistry
STD NMR results suggest that naphthyl moiety adopts The build-up curves are fitted to the monoexponential Eq. 2,
View Article Online
DOI: 10.1039/C5OB01916H
conformation where Hb proton (orange on Fig. 10) is the where STD-AF stands for the STD amplification factor of a
closest to the receptor followed by Hc proton (yellow on Fig. given proton at saturation time t, STDmax is the maximal STD
10). Weakest interaction and thus greatest receptor-ligand intensity obtainable when long saturation times are used, and
interproton distance was observed for He proton, marked with ksat is the observed saturation rate constant.
blue. On the other hand, flexibility resulting from glycerol
(Eq. 2)
linker allowed unforeseen engagement of phenyl group in
more polar area, where it forms hydrogen bond with Asn344
and contacts with Arg345 (possible cation- interaction) of DC-
The hyperbolic behaviour of the curves obtained from the plot
of STD-AF as a function of ligand concentration is appropriately
described by Eq. 3, where STD-AF is a STD amplification factor,
αSTD represents the maximum amplification factor, [L] is a
ligand concentration and KD is dissociation constant.
SIGN. Conformation of phenyl entity thus apparently depends
on quite prominent Hf proton-receptor proximity and
formation of hydrogen bond between esterified carboxylic
group and Asn344 side chain. Observation of phenyl moiety
retention in polar environment of the binding site offers new
insights in ligand behaviour within flat binding sites and
additionally emphasizing the need for further designing of
novel and more potent DC-SIGN antagonists.
(Eq. 3)
For the non-linear least-squares curve-fitting to the Eq. 2 and
Eq.3 was used Origin 8.1 software.33 All experimental data
were presented with standard errors bars.
Experimental
Molecular modelling
Chemistry.
Computer hardware and software. All the computational work
was performed on a workstation with four octal core AMD
Opteron CPU processors, 16 GB RAM, two 750 GB hard drives,
running 64-bit Scientific Linux 6.0.
Detailed information is given in the Supporting information.
STD experiments. STD NMR samples were prepared in 25 mM
TRIS-d11 (pD = 8.0), 150 mM NaCl, 4 mM CaCl2 in 2H2O and 25%
of DMSO-d6. In all STD experiments the concentration of
receptor DC-SIGN was 8 μM. DC-SIGN ECD protein (residues
66-404) were overexpressed and purified according to the
already published procedure.20, 22, 32 For the STD NMR studies,
a psevdo-2D version of STD NMR sequence with DPFGSE water
suppression was used for the interleave acquisition of on- and
off-resonance spectra at 25 °C with 512 scans. For selective
saturation cascades of Gaussian pulses with a length of 50 ms
and 10 dB spaced by 0.1 ms delays were employed. The on-
resonance irradiation was performed at 0.9 ppm; the off-
resonance frequency was 30 ppm. For the protein signal
suppression was used 30 ms spin lock filter. The STD spectrum
was obtained by subtraction of saturated spectra from
reference spectra. The fractional STD effect was calculated by
(I0-Isat)/I0, where Ioff stands for intensity of signal in off-
resonance spectrum. Isat is intensity of signal in on-resonance
spectrum. Ioff -Isat represents the intensity of the STD NMR
spectrum. The appropriate blank experiments, in the absence
of the protein, were performed to assure the absence of direct
irradiation of the ligand. Saturation times to obtain the STD
build-up curves were 0.1, 0.3, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5,
3.0, 4.0, 5.0 s. In titration study, five STD experiments were
performed with varying concentrations, 80, 240, 400, 560 and
800 μM.
Ligand and protein preparation. The three-dimensional
models of compounds 1-4 were built in ChemBio3D Ultra
13.0.34 The geometries and charges of the molecules were
optimized using the MMFF9435 force field and partial atomic
charges assigned in ChemBio3D Ultra 13.0. The energy was
minimized until the gradient value was smaller than 0.001
kcal/ (mol×Å). The optimized structure was further refined
with GAMESS interface in ChemBio3D Ultra 13.0, using the
semi-empirical PM3 method, QA optimization algorithm and
Gasteiger Hückel charges for all atoms for 100 steps.
The crystal structure of DC-SIGN CRD in complex with
tetramannoside Man4 (PDB entry: 1SL4) was taken as a
receptor for docking calculations using FlexX36, 37 as available in
LeadIT.38 Receptor was prepared in LeadIT graphical user
interface using the Receptor wizard. Amino acid residues
within a radius of 7 Å around Man4 were defined as the
binding site. Hydrogen atoms were added to the binding site
residues and correct tautomers and protonation states were
assigned. Water molecules and the ligand were deleted from
the crystal structure, while Ca2+ ion in the Ca2+ binding site was
defined as part of the receptor. Pharmacophore type
constraints in FlexX-Pharm were defined in order to correctly
place the core mannose residue during the docking procedure.
First, PharmMetal pharmacophore with spherical coordination
around Ca2+ ion was defined to account correctly for complex
interactions between Ca2+ ion (406) and D-mannose residue of
the designed ligands. Secondly, side chain carboxylate groups
of Glu347 and Glu354 were defined as hydrogen bond
acceptors, which form hydrogen bonds with 3- and 4-hydroxyl
groups of D-mannose residue in the crystal structure. All three
selected pharmacophore type constraints were specified as
essential.
STD-amplification factor is determined according to Eq. 1, in
which is a STD amplification factor. Ioff stands for
intensity of signal in off-resonance spectrum. Isat is intensity of
signal in on-resonance spectrum. Ioff -Isat represents the
intensity of the STD NMR spectrum. [L] is the concentration of
ligand and [P] stands for the concentration of receptor.
(Eq. 1)
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For docking with FRED software (Release 3.0.1, OpenEye using SHAKE algorithm.48 The simulation consisVtieewdAortifcletOhnrleinee
DOI: 10.1039/C5OB01916H
USA; consecutive steps: (i) solvent equilibration for 0.5 ns with
Scientific
Software,
Inc.,
Santa
Fe,
NM,
www.eyesopen.com), the protein was prepared using MAKE ligand and protein constrained harmonically around the initial
RECEPTOR (Release 3.0.1, OpenEye Scientific Software, Inc., structure; (ii) equilibration of the complete system for 1 ns
Santa Fe, NM, USA; www.eyesopen.com).28-30 We determined with ligand and protein released; (iii) an unconstrained 10 ns
the grid box size; inner and outer contours of the grid box production run to allow the protein and the ligand to position
were calculated automatically. The outer contour size was themselves according to physical forces between them. The
6195 Å3, while the grid box size was 9216 Å3. The setup of trajectory of the equilibration and production run was used for
contours was set as “Balanced”. Amino acid residues Asn365, analysis in VMD. The MD simulation of each complex was
Glu347 and Glu354 and Ca2+ ion were defined as repeated three times.
pharmacophore type constraints. FRED requires the
preparation of input conformers of each ligand prior to
Conclusions
docking. These conformers were generated using OMEGA
2.4.639 from OpenEye Scientific Software.
In the present study we investigated the interactions between
Ligand docking. The FlexX molecular docking program, as
available in LeadIT, was used for ligand docking. Hybrid
algorithm (enthalpy and entropy driven ligand binding) was
used to place the ‘base fragment’. The Maximum number of
solutions per iteration and the Maximum number of solutions
per fragmentation parameter values were increased to 1000,
while other parameters were set at their default values.
Proposed binding modes and scoring function scores of the top
ten highest scored docking poses per ligand were evaluated
and the highest ranked binding pose was used for graphical
representation in PyMOL.
Ligands 1-4 were also docked using FRED (version 3.0.1.
OpenEye Scientific Software, Inc., Santa Fe, NM, USA;
www.eyesopen.com).28-30 Docking resolution was set to high,
other settings were set as default. Twenty-five docking
solutions were inspected visually and the best ranked FRED-
calculated conformation was used for analysis and
representation.
four monovalent mannose-based ligands and extracellular
domain of DC-SIGN. Studied ligands, some of which are novel
entities, consist of α-D-mannose connected by glycerol linker
to different aryl moieties, which were expected to tune
interactions of ligands in hydrophobic groove of the receptor.
The combination of saturation transfer difference (STD) NMR
spectroscopy and molecular modelling has been used to
examine binding affinity of monovalent glycomimetics for DC-
SIGN at the atomic level of resolution. Our study showed that
four α-D-mannoside ligands interact with the binding site
predominantly with mannose moiety. Importantly, the other
two aromatic groups that are bound to α-D-mannose through
glycerol linker demonstrate interaction that can be related to
their substituent pattern. Ligand with naphthyl and meta-
substituted phenyl ring exhibited the most favourable binding
characteristics, which might imply involvement of new
contacts of substituted phenyl moiety in more polar area of
flat binding site of DC-SIGN. Although our first impression was
that carboxylate in the studied ligands should interact
primarily with Arg345, it seems that the Asn344 residue forms
H-bond with the polar group and thus dictates the main
interactions with the polar substituent at phenyl ring, while
Molecular dynamics. The molecular dynamics package NAMD
(version 2.9)40 and CHARMM22 force field41 were used for
molecular dynamics simulations of DC-SIGN–ligand complexes.
The FlexX best scored docking pose of compounds (R)-
in complex with DC-SIGN CRD was used as the initial
structure for MD simulation. Molecular mechanics parameters
for compounds (R)- and (S)-3 were estimated using
3 or (S)-
3
Arg345 possibly forms cation-
moiety. Our findings point that besides lipophilic residues,
possible involvement of H-bonds and /or cation- might boost
 interaction with the phenyl
3

ParamChem tool.42-44 Steepest descent (10 000 steps) and
adopted basis Newton–Raphson (10 000 steps) energy
minimizations were first performed to remove atomic clashes
and to optimize the atomic coordinates of the protein – ligand
complex. The structure of the energy minimized complex for
MD simulation was prepared using psfgen in VMD (version
1.9.1.).45 The complex was then embedded in a box of water,
which was modelled explicitly by a TIP3P model.46 The system
was neutralized by addition of KCl at 0.4 M concentration. The
MD simulation was carried out in the NPT ensemble employing
periodic boundary conditions. Langevin dynamics and Langevin
piston methods were used for temperature (300 K) and
pressure (1 atm) control, respectively. Short- and long-range
forces were calculated every 1 and 2 timesteps, respectively,
with a time step of 2.0 ps. The smooth particle mesh Ewald
method47 was used to calculate electrostatic interactions. The
short-range interactions were cut off at 12 Å. All chemical
bonds between hydrogen and heavy atoms were held fixed
the interaction of small molecule DC-SIGN ligands and thus
offer new possibilities in further designing of novel, stronger
DC-SIGN antagonists.
Acknowledgements
This work was supported by the Slovenian Research Agency
(ARRS, grants P1-0208, P1-0242 and J1-6733). The authors
thank Matjaž Weiss for the help in synthetic laboratory and
Prof. Dr. Franck Fieschi for the DC-SIGN ECD produced in his
laboratory.
Notes and references
1
M. Altfeld, L. Fadda, D. Frleta and N. Bhardwaj, Nat. Rev.
Immunol., 2011, 11, 176.
H. Liu, W. Bi, Q. Wang, L. Lu and S. Jiang, Biomed. Res. Int.,
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2
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Organic & Biomolecular Chemistry
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Organic & Biomolecular Chemistry
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