Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8

Article abstract of DOI:10.1002/cmdc.201800224

Galectin-8 is a β-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-β-d-galacto

Full text of DOI:10.1002/cmdc.201800224

Accepted Article
Title: Structure-based Design of a Monosaccharide Ligand Targeting
Galectin-8
Authors: Mohammad Bohari, Xing Yu, Chandan Kishor, Brijesh Patel,
Rob-Marc Go, Hadieh Alsadat Eslampanah Seyedi, Yaron
Vinik, Irwin Darren Grice, Yehiel Zick, and Helen Blanchard
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To be cited as: ChemMedChem 10.1002/cmdc.201800224
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10.1002/cmdc.201800224
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Structure-based Design of a Monosaccharide Ligand Targeting
Galectin-8
Mohammad H. Bohari [a], Xing Yu [a], Chandan Kishor [a], Brijesh Patel [a], Rob Marc Go [a], Hadieh A.
Eslampanah Seyedi [a], Yaron Vinik [b], I. Darren Grice [a,c], Yehiel Zick [b], and Helen Blanchard [a]*
[a]
Dr. M. H. Bohari (ORCID: 0000-0001-9375-5290), Dr. X. Yu, Dr. C. Kishor, B. Patel, R. M. Go, H. A. E. Seyedi,
Prof. H. Blanchard (ORCID: 0000-0003-3372-5027)
Institute for Glycomics
Griffith University
Parkland’s Drive, Gold Coast Campus 4222, Australia
E-mail: h.blanchard@griffith.edu.au
[b]
Dr. Y. Vinik, Prof. Y. Zick
Department of Molecular Cell Biology,
Weizmann Institute of Science
Herzl St 234, Rehovot, Israel.
[c]
Dr. I. D. Grice (ORCID: 0000-0001-9218-457X)
School of Medical Science
Griffith University
Parkland’s Drive, Gold Coast Campus, 4222, Australia.
Supporting information for this article is given via a link at the end of the document.
Abstract: Galectin-8 is a β-galactoside-recognising protein that has
a role in the regulation of bone remodelling and is an emerging new
target for tackling diseases with associated bone-loss. We have
designed and synthesised methyl 3-O-[1-carboxyethyl]-β-D-
galactopyranoside (compound 6) as a ligand to target the N-terminal
domain of galectin-8 (galectin-8N). Our design involved molecular
dynamics (MD) simulations that predicted 6 to mimic the interactions
made by the galactose ring as well as the carboxylic acid group of
3′-O-sialylated lactose (3′-SiaLac), with galectin-8N. Isothermal
titration calorimetry (ITC) determined that the binding affinity of
galectin-8N for 6 was 32.8 μM, while no significant affinity was
detected for the C-terminal domain of galectin-8 (galectin-8C). The
crystal structure of the galectin-8N-6 complex validated the predicted
binding conformation and revealed the exact protein-ligand
interactions that involve galectin evolutionarily conserved amino
acids and also those unique to galectin-8N for recognition. Overall,
disorders through the regulation of T-cell homoeostasis [4] and is
critically involved in capillary tube formation and angiogenesis [5]
.
Antibacterial activity, mediated through induction of selective
autophagy, highlights an additional cellular mechanism to
combat infection recruiting galectin-8 [6]. It is of interest that
galectin-8 has shown in vivo regulation of bone remodelling via
enhancing the expression of bone resorbing factors that are
attributed to increased bone turnover culminating in reduced
A)
B)
S6
Glu89
Asn79
S5
Arg69
S4
Primary
binding site
Asn67
His65
S3
Arg45
Trp86
Extended
Trp86
binding site
S2
Gln47
S1
Arg59
Tyr141
we have initiated and demonstrated
a rational ligand design
campaign to develop a monosaccharide-based scaffold as a binder
of galectin-8.
S3-S4 loop
Figure 1. Overview of the galectin-8N carbohydrate recognition
domain. a) The CRD showing the carbohydrate binding face of the “jelly-
roll”. The primary and the extended binding site are indicated. b) Binding
conformation and hydrogen bond and salt-bridge interactions (in grey
dashed lines) made by 3′-SiaLac (in sticks; carbon in black, oxygen in
red) upon binding to galectin-8N residues (in sticks; carbon in green,
[8a]
oxygen in red and nitrogen in blue) PDB ID: 3AP7
.
bone mass [7]. Inhibition of galectin-8 may thus offer a potential
new therapeutic approach in managing diseases with bone-loss.
Available structural information of galectin-8N bound to different
biologically relevant oligosaccharides provide insight into various
biological processes and highlights key interactions imparting
affinity and specificity to a ligand [8]. Typically, the CRD has a b-
sandwich “jelly-roll” topology formed from two β-sheets. The
concave surface of the roll that constitutes the carbohydrate-
binding site, comprises six β-strands, S1-S6 (Figure 1a). The
galactose recognition site consists of evolutionarily conserved
amino acids on strands S4-S6. Galectin-8N preferentially
Introduction
Galectin-8 is a β-galactoside recognising protein that contains
two carbohydrate recognition domains (CRD) in tandem, linked
by a variable length amino acid linker [1]. It has widespread
tissue distribution in both normal and tumour cells. Within the
cell, galectin-8 occurs in the nucleus, the cytoplasm, and also it
is secreted into the extracellular space [2]. Apart from being
involved in cell-to-cell and cell-to-surrounding communication,
an increasingly broader functional spectrum of galectin-8 is
apparent [3]. Galectin-8 plays an important role in inflammatory
1
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10.1002/cmdc.201800224
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Results and Discussion
recognises anionic oligosaccharides such as 3′-sulfated lactose
and 3′-O-sialylated lactose (3′-SiaLac) [9]. This preferential
binding arises from the presence of unique structural features in
the galectin-8N CRD such as a long S3-S4 loop, bearing an
arginine residue (Arg59), and a glutamine (Gln47) on strand S3
[8a]. The crystal structure of 3′-SiaLac bound to galectin-8N
shows engagement of the carboxylic acid group of sialic acid in
salt bridge interactions with Arg59 and hydrogen bonding to
Ligand design hypothesis though MD simulations
MD simulations were performed to analyse the relative strength
and propensity of binding interactions observed in the crystal
structures, as well as for detection of possible alternate
interactions assisting in conceptualising further our ligand design
hypothesis. Lactose as the native galectin ligand, was also
included (galectin-8N-lactose complex PDB ID: 5T7S [8b]) in the
MD analysis along with the galectin-8N-3′-SiaLac crystal
structure (PDB ID: 3AP7 [8a]). Hydrogen bond occupancy
analysis was performed to examine the longevity of interactions
observed in the crystal structure. The hydrogen bonds observed
in galectin-8N-lactose and galectin-8N-3′-SiaLac crystal
structures were found with occupancy of 60-100 % (total
occupancy for the residue) during simulations (Figure 3). An
identical interaction profile for the common lactose moiety within
both ligands was observed for the conserved residues His65,
Glu89, Arg69, and Asn79 (Figure 3). The occupancy for Arg45
hydrogen bonds was higher in 3′-SiaLac compared to that in the
lactose complex. The carboxylate group of 3′-SiaLac showed
100 % occupancy of salt bridge interactions with Arg59 and over
60 % occupancy of hydrogen bond with Gln47 and Trp86. Our
simulation of galectin-8N-3′-SiaLac crystal complex revealed that
60 % of the time the Gln47 side chain was re-orientated by 180°
interchanging the Nd1 and Od1 compared to the deposited PDB
coordinates (PDB ID: 3AP7[8a]) and thus enabled hydrogen
bonding to occur with the carboxylic acid group. Critically, amino
acids Arg59 and Gln47 are unique to galectin-8N and are
identified as supposed ligand specificity hotspots [8]. Taking
advantage of this existing structural information and findings
from our in silico virtual screening (not repoted here) a
monosaccharide-based ligand of galectin-8N was conceived to
exploit interactions with both unique and conserved residues of
the galectin-8N binding site.
Figure 2. The ligand design concept. Depicted are the chemical structures
of 3′-SiaLac, the designed compound 6, and the surrounding amino acid
residues in the galectin-8N binding site. The interaction with Arg59 and
Gln47 (labelled in red) are the unique residues that 6 is designed to exploit.
Gln47, and the pyranose ring of the galactose formed conserved
interactions that are observed for the counterpart of lactose
(Figure 1b) [8a]. These interactions between the anionic group of
the glycans with Arg59 and Gln47 were attributed as the origin
of the high affinity towards galectin-8N [8a]
.
Given the inherent nature of galectins to recognise β-
galactoside-containing glycans, and galectin-8N’s preferential
recognition of anionic sugars, then a monosaccharide-based
ligand such as galactose bearing
a
negatively charged
250
225
200
175
150
125
100
75
carboxylic acid group at the 3′-position akin to 3′-SiaLac seemed
promising as a ligand scaffold. Incorporation of a carboxylic acid
group in ligands to engage unique binding site residues of
galectin-8N was also supported by our in silico virtual screening
of a library of compounds with a diverse set of functional groups
where 25% of the top hits identified had a carboxylic acid group
(unpublished). The mammalian galectins have wide ranging
biological functions but exhibit a shared primary binding site and
hence exploiting unique residue/s in close proximity of this site
is vital in achieving galectin specificity. We report the first
50
25
Figure 3. Interaction analysis from 100 ns MD simulations. Percentage
0
of frames with specific hydrogen bonds between the galectin-8N binding site
residues and the ligand (3′-SiaLac, lactose (Lac) and 6) atoms. For clarity,
the hydrogen bond occupancy with respect to individual residues is
Lac
6
3′-SiaLac
represented.
synthetic
ligand
(methyl
3-O-[1-carboxyethyl]
-β-D-
The stable interactions made by the galactose ring and the
carboxylic acid group in the galectin-8N-3′-SiaLac crystal
complex were incorporated in the design of 6 (Figure 2). The
main advantage for 6 being the anticipated specific recognition
by galectin-8N, and associated increase in binding affinity and
specificity, by the carboxylic acid group on the 3′-position of the
galactose. Furthermore, 6 can be synthesised with relative ease
and has increased stability over 3′-SiaLac. Lactose (molecule’s
atomic coordinates) from the galectin-8N-lactose complex (PDB
ID: 5T7S [8b]) was modified to obtain the initial placement of 6 in
the galectin-8N binding site (Supplementary Figure S1). This in
silico galectin-8N-6 complex was subjected to 100 ns simulation
galactopyranoside, see Figure 2) (6) developed through a
structure-based rational design approach targeting galectin-8
(specifically the N-terminal CRD). We demonstrate its ability to
bind with affinity at the micromolar range to a monosaccharide-
based scaffold, as well as its preference towards the galectin-8N
domain.
2
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to investigate the feasibility of the interactions observed in the
initial model and the suitability of 6 in the binding site.
Compound 6 stayed in the primary binding site for the duration
of the simulation, retaining the typical CH-π interactions with the
evolutionarily conserved Trp86, as observed for the natural
galectin ligands. Hydrogen bond occupancy analysis revealed
an almost identical interaction profile of 6 to that observed for
the corresponding part of the 3′-SiaLac in the galectin-8N-3′-
SiaLac simulation (Figure 3). The hydrogen bond occupancy,
particularly for the unique residues Arg59 and Gln47 and the
conserved residue Trp86, noted for 6 is identical to that
observed for 3′-SiaLac. The MD interaction analysis suggests
that the galactose ring of 6 would occupy the primary binding
site of galectin-8N and that the carboxylic acid group at the 3′-
position would engage in interactions with the unique Arg59 and
Gln47. Based on these predictions, we synthesised 6, and its
binding affinity, binding mode and interactions to galectin-8N
were validated using ITC and X-ray crystallography.
Validation of interactions by X-ray crystal structure
determination
We subsequently performed crystallographic analysis to
investigate the binding mode and interactions of 6 to galectin-
8N. The galectin-8N-6 complex was obtained by soaking the
ligand into the apo galectin-8N crystals, and the structure then
determined at 2.1 Å resolution. The electron density maps reveal
unambiguous placement of 6 in the galectin-8N binding site
(Figure 4, Supplementary Figure S2). A clear protrusion in the
difference electron density map pointing towards the conserved
Arg69 confirmed the positioning of the O4′ hydroxyl of the
galactose. Further, the planar topology of the electron density
adjacent to the unique Arg59 is consistent with the placement of
the carboxylic acid group and thereby confirming the overall
placement of 6 (Figure 4). The electron density for the methyl
group of the propionic acid side chain that points toward solvent
is weak. However, it is sufficient to give the direction of methyl
group, correlating with an R-configuration at the carbon chiral
centre. The positive difference electron density that appears as
an extension from the anomeric methoxy group, is concluded to
be a water molecule.
Chemical synthesis of the designed ligand
A)
B)
Glu89
Arg69
Asn79
Asn67
Arg45
Trp86
Arg59
Trp86
His65
Gln47
Arg59
Scheme 1. Synthesis scheme for 6. Reagents and condition: a) CSA,
benzaldehyde dimethyl acetal, ACN, 60ºC, 2.5 h (yield 88%); b) Ag2O
(freshly prepared), AcCl, KI, DCM, RT, 16 h (yield 70%); c) DIPEA, MOMBr,
DCM, reflux, 16 h (yield 80%); d) Na metal, MeOH, RT, 1.5 h (yield 90%); e)
NaH, 2-chloropropionic acid, anhydrous 1,4-dioxane, 50 ᵒC, 36 h (yield
60%); f) concentrated HCl, MeOH (yield 75 %).
Figure 4. Galectin-8N-6 complex. A) Electron density (blue mesh) 2|Fo|-
|Fc| αc contoured at 1σ, for 6 (in sticks; carbon in black, oxygen in red) in
complex with galectin-8N. B) Hydrogen bonding and salt-bridge interactions
(in grey dashed line) made between 6 and galectin-8N (in sticks; carbon in
green; oxygen in red; nitrogen in blue).
Synthesis of 6 was initiated with β-D-methyl galactoside as the
starting material (Scheme 1). The C4-OH and C6-OH group of
the β-D-methyl galactoside was 4,6-benzylidene protected (1)
utilising benzaldehyde dimethyl acetal with a catalytic amount of
camphor sulfonic acid (CSA)[10] to afford 1. Following this, 1
underwent selective 3O-acetylation using silver oxide (Ag2O),
acetyl chloride (AcCl) and a catalytic amount of potassium iodide
(KI)[11] to give 2. The acetylated galactoside 2 was then 2O-
methoxymethyl ether (MOM) protected using an adapted
procedure [12], employing diisopropyl ethyl amine (DIPEA) in
dichloromethane (DCM) to give 3. After quantitative
deacetylation using sodium metal in methanol to give 4, the 2-
chloropropionic acid side chain was coupled using sodium
hydride (NaH) in 1,4-dioxane to yield 5 [13]. A final deprotection at
Overall, the binding mode observed for the galactose portion of
6 is identical to that noted for the corresponding part of the
galectin-8N-lactose complex [8b]. The O4′ of the galactose
engages in hydrogen bonding with His65, Asn67, Arg45, Arg69
whereas the O6′ hydrogen bonds with Asn79, and Glu89, as
also noted from our simulations (Figure 4). Importantly, from the
original design concept, the carboxylic acid was found in a
geometrically favoured position to make ionic interactions with
the unique Arg59 and hydrogen bonding interactions with Gln47.
Furthermore, the placement of the anionic group and the
galactose ring is identical to the equivalent part of 3′-SiaLac
complexed with galectin-8N (Supplementary Figure S3). The
carboxylic acid group displaced a water molecule that was
observed in the vicinity of the conserved Trp86 and Arg59 in the
galectin-8N-lactose complex (PDB ID: 5T7S [8b]), suggesting
overall stronger binding. With the galectin-8N-6 crystal structure,
we validated our design concept and the predicted binding
conformation for 6.
positions 2- and 4,6-positions was performed using concentrated
[14]
HCl in methanol, to yield a racemic mixture of compound 6
.
The structure of 6 was confirmed with the 13C NMR signature
chemical shifts for two methyl groups being observed, one
consistent with the anomeric at 55.84 ppm and the other for the
propionic acid side chain at 17.63 ppm. Additionally,
1
corresponding H NMR peaks were observed and a molecular
ion from MS confirmed the integrity of the ligand.
3
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extended binding site would play a critical role in recognising 6.
We used the crystallographic conformation of 6 from the
galectin-8N-6 complex (PDB ID: 5VWG) and generated the in
silico galectin-8C-6 complex model through superimposition of
the two CRDs. The in silico galectin-8C-6 complex model
reveals possible hydrogen bonding interactions of 6 with His271
(His65 in galectin-8N), Arg275 (Arg69), Glu294 (Glu89), and
Asn284 (Asn79) as observed in the galectin-8N-6 crystal
structure (Figure 5). However, the carboxylic acid side chain of 6
lacked interactions with the galectin-8C binding site due to the
absence of Arg59 on S3-S4 loop, the presence of Ser255 in
place of Arg45, and Asn257 in place of Gln47 (Figure 5). The
favourability and stability of ligand binding were analysed
from100 ns MD simulations, while binding free energies were
estimated using the MMPBSA method. Compound 6 occupied
the primary binding site in galectin-8N during the length of
simulation, with no significant fluctuations in the ligand
placement detected (Figure 5). However, 6 showed significant
fluctuations in case of the galectin-8C binding site, possibly due
to lack of the long S3-S4 loop bearing unique residue like Arg59
(Figure 5). The average estimated ligand binding free energy for
the galectin-8C-6 complex was therefore observed to be only
half (-25.5 kcal/mol) that estimated for the galectin-8N-6
complex (-60.6 kcal/mol). Overall, our simulation results and
binding free energy analysis suggest favourability of the
Table 1. Crystallographic data merging and refinement statistics for galectin-
8N-6 complex structure.
Data
Galectin-8N-6
Indexing
Crystal system
Space group
Unit cell
Orthorhombic
P212121
a=45.72, b=50.32, c=80.87
Merging and Scaling
Resolution (Å)
Total observations
Unique observations
Multiplicity
42.73 – 2.10
54110 (4147) a
11126 (873)
4.9 (4.8)
Completeness (%)
I/σ
98.0 (95.0)
5.9 (1.8)
Rmerge (%)
Refinement
14.9 (78.2)
Resolution
42.59-2.10
22.2
designed compound
6 towards galectin-8N compared to
Rfactor (%)
galectin-8C.
Rfree (%)
25.1
Number of atoms
Protein
1196
18
Ligand
Water
103
Root mean square deviation
Bond length (Å)
Bond angle (ᵒ)
Ramachandran plot statistics
Favoured (%)
Allowed (%)
Average B-factor (Å2)
Protein
0.007
1.29
97.2
2.8
26.3
Ligand
36.3
Figure 5: Binding mode comparison of 6 towards the galectin-8N
(crystal structure [PDB ID: 5VWG]; on the left) and the galectin-8C (in
silico complex; on the right). On top, key binding site residues are
represented in sticks (carbon in green for protein atoms and carbon in black
for ligand atoms) and hydrogen bonds and salt-bridge in grey dashed lines.
Bottom figures show overlayed ligand (in the wire; carbon in black and
oxygen in red) coordinates extracted from the 100 ns simulation bound to
galectin-8N (bottom right) and galectin-8C (bottom left). The MMPBSA
estimated binding free energy (in kcal/mol) is highlighted in yellow.
Water
33.0
PDB ID
5VWG
[a] The value in parenthesis is for the highest-resolution shell.
Binding specificity through MD simulations
Assessing the preference of 6 towards the CRDs of galectin-8,
we have employed MD simulations to predict and compare its
binding mode and interactions with galectin-8N and galectin-8C.
The primary binding site of the two CRDs of the galectin-8 is
mostly conserved. However, amino acid differences in the
Binding affinity determination and specificity by ITC
Binding affinities (Kd) of compound 6, lactose and 3′-SiaLac
towards galectin-8N and galectin-8C were determined using ITC.
In the experiment, heat changes occurring from the ligand’s
4
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titration into a protein solution and the ligand’s titration into a
agreement with the previously reported affinities for lactose and
3′-SiaLac towards galectin-8N and galectin-8C (Table 2). The
[9]
buffer (without the protein) were recorded. Compound
6
exhibited 1:1 stoichiometric binding to galectin-8N with a binding
affinity of 32.8 ± 1.8 μM, whilst that determined for 3′-SiaLac was
2.3 ± 0.2 μM (Figure 6 and Supplementary Figure S4). The
binding affinity of the monosaccharide 6 was slightly stronger
than the measured affinity for the disaccharide lactose (47.4 ±
1.1 μM), indicating the overall efficiency of the designed ligand.
Interestingly, 6 did not show significant binding towards galectin-
8C. This is presumed to be mainly due to the absence of Arg59
on the S3-S4 loop, supporting the observed difference in the
estimated binding free energies from MD simulations. The
discrepancy in the absolute Kd values previously reported for
lactose (1.7-3.1 μM) and 3′-SiaLac (53 nM) using the
fluorescence anisotropy assay could potentially be due to the
difference in the principle of detection and variation in
experimental parameters such as buffer, pH and temperature [15]
.
Table 2. Binding affinity and estimated thermodynamic parameters from ITC
experiments.
Kd
ΔH
-TΔS
ΔG
Ligand
n
(μM)
(kJ/mol)
(kJ/mol)
(kJ/mol)
-6.4
±1.3
0.92
±0.04
Lactose
47.4 ±1.1
2.3 ±0.2
32.8 ±2.1
331.1
-18.3
20.5
-9.3
-19.2
-
-24.6
-32.1
-25.6
-19.8
3′-
SiaLac
-52.7
±0.8
0.81
±0.01
-16.3
±1.3
0.77
±0.04
Cpd 6
-0.6
0.6
±
1.28
±0.28
Lactose
3′-
SiaLac
>1000
-
-
-
-
No
Cpd 6
-
binding
Conclusions
Overall, we have employed a structure-based approach to
rationally design a monosaccharide-scaffold ligand to target
galectin-8N. The preference of anionic saccharides towards
[9, 15]
galectin-8N
and the results of our in silico virtual screening
suggested the favourability of anionic groups in the galectin-8N
binding site. Considering previously obtained in silico data, the
most frequent hydrogen bonding interactions exhibited during
MD simulations, the synthetic feasibility and relative stability
over 3′-SiaLac, 6 was designed. The ligand design hypothesis
was taken experimentally validated by synthesising 6 and
evaluating its binding to galectin-8N through X-ray
crystallography and ITC. The X-ray structure of the galectin-8N-
6 complex validated our predicted conformation, where the
ligand explored both the evolutionarily conserved and the unique
amino acids of galectin-8N for interaction. The interaction profile
observed for 6 was identical to the corresponding part of native
ligand lactose, and the template molecule 3-SiaLac when bound
to galectin-8N. The binding specificity of 6 was addressed by
determining its binding affinity using MD simulations and ITC
towards the two CRDs of galectin-8. The ITC determined binding
data suggests preferential binding of compound 6 to galectin-8N
(32.8 μM) over galectin-8C (no binding detected), and its binding
affinity was slightly better than the affinity of lactose (47.4 μM).
This favourability was also indicated from our MD simulations
and was most likely the result of our targeted exploitation of
unique residues for interactions by 6. The promising binding
affinity of our monosaccharide-based 6 over the disaccharide
Figure 6. Isothermal calorimetric analysis. Binding isotherm for
titration of 1mM of 6. A) with 200 mM galectin-8N and B) 200 μM
galectin-8C in Tris buffer at pH 8 containing 100 mM NaCl and 4 mM
BME. Lactose and 3′-SiaLac ITC graphs are presented in
supplementary data (Figure S4).
affinity of galectin-8C for lactose was 331.1 μM, whilst no
significant binding was detected for 3′-SiaLac, as also previously
reported [9]. These findings suggest the binding preference of 6
towards galectin-8N over galectin-8C. The results with
compound 6 thus support our design hypothesis of exploring the
unique residue Arg59 found in galectin-8N for gaining affinity
along with specificity. Overall, our binding affinity data is in good
5
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dichloromethane (DCM):methanol (MeOH) to yield 1 (88%). The product
was consistent with the reported title compound [10]. 1H NMR (CDCl3, 400
MHz): δ = 3.52 (1H, s), 3.59 (3H, s, OCH3), 4.05 (1H, dd, J=7.6, 11.6 Hz),
4.13 (1H, dd, J=1.6, 12.4 Hz), 4.35 (1H, d, J=7.6 Hz), 4.41 (1H, dd, J=1.6,
12.4 Hz), 4.46 (1H, dd, J=0.8, 3.6 Hz), 4.85 (1H, dd, J=3.68, 11.6 Hz),
5.50 (1H, s, PhCHO2), 7.35 (3H, m, ArH), 7.49 (2H, m, ArH).
lactose is indicative of the overall improved ligand efficiency.
Furthermore, this prompt for exploration of other functional
groups on the galactose core towards identifying novel
compounds as galectin-8N ligands and potential inhibitors. Our
rational ligand-design campaign has led to successful
identification of a monosaccharide-based scaffold that binds to
galectin-8N and forms a proof-of-concept study. This scaffold
provides a basis for further optimisation aimed at enhancing
binding affinity and specificity towards galectin-8 with the aimed
application as a therapeutic in managing diseases that have
associated bone-loss.
[11]
Methyl 3-O-acetyl 4,6-O-benzilidene-β-D-galactopyranoside (2)
:
Compound 1 was dissolved in anhydrous DCM, cooled to -20 ᵒC using
[29]
an ice-salt mixture. Freshly prepared silver oxide (Ag2O)
was added
and left for 30 minutes with stirring, followed by slow addition of acetyl
chloride (AcCl) and KI [11]. The reaction was left stirring overnight at room
temperature. Ag2O was removed via filtration, and the solvent was
removed in vacuo. The product was purified by flash chromatography
(1:1 hexane:ethyl acetate (EtOAc)) to give 2 (70% yield). 1H NMR (CDCl3,
400 MHz) δ = 2.11 (3H, s, OAc), 2.52 (1H, brs), 3.48 (1H, d, J=1.5 Hz),
3.56 (3H, s, OCH3), 3.93 (1H, dd, J= 1.6, 8.0), 4.01 (1H, m, J=2.0, 12.4
Hz), 4.21 (1H, d, J=8.0 Hz, H1), 4.26 (1H, dd, J=1.6, 12.4 Hz), 4.32 (1H,
dd, J=0.8, 3.6 Hz), 4.82 (1H, dd, J=3.6, 10.2 Hz), 5.48 (1H, s, OCHPh),
7.32-7.37 (3H, m, ArH), 7.47-7.50 (2H, m, ArH).
Experimental Section
Molecular dynamics simulations
The initial coordinates of the designed compound 6 bound to galectin-8N
were obtained by modifying the lactose from the galectin-8N-lactose
complex (5T7S [8b]) using the BIOVIA Discovery Studio Visualiser [16]. In
Methyl
2-O-methoxymethyl-3-O-acetyl-4,6-O-benzilidene-β-D-
silico galectin-8C-6 complex was generated by removing the bound
galactopyranoside (3): The methoxy methyl (MOM) ether protection
procedure was adapted from literature [12]. Compound 2 was dissolved in
DCM under argon at room temperature, diisopropyl ethylamine (DIPEA)
was added at 0ᵒC followed by drop-wise addition of bromomethyl methyl
ether and refluxed overnight. The reaction was diluted with DCM and
washed with water and brine solution, then purified using flash column
chromatography (2:1 hexane:EtOAc) to give 3 (80% yield). 1H NMR
(CDCl3, 400 MHz): δ 2.10 (3H, s, OAc), 3.38 (3H, s, MOM), 3.47 (1H, d,
J=1.1 Hz), 3.55 (3H, s, OCH3), 3.95 (1H, q, J=2.4, 10.2 Hz), 4.04 (1H, dd,
J=1.8, 12.4 Hz), 4.31 (1H, dd, J=1.5 Hz), 4.33 (1H, d, J=2.2 Hz), 4.35 (1H,
dd, J=0.7, 3.7 Hz), 4.68 (1H, d, J=6.4 Hz, H1), 4.85 (2H, m, MOM), 5.48
(1H, s, CHPh), 7.35 (3H, m, ArH), 7.50 (2H, m, ArH). 13C NMR (CDCl3,
100 MHz): δ = 21.1, 55.7, 56.9, 66.1, 69.0, 72.7, 73.5, 97.2, 101.1, 104.1,
126.4, 128.1, 129.0, 137.7, 170.8. MS (ESI): m/z calculated for
C18H24NaO8 [M+Na]+ 391.2, found 391.2.
[17]
peptide and superimposing the galectin-8C crystal structure (4GXL
)
on to the galectin-8N-6 crystal structure using the MatchMaker utility of
[18]
UCSF Chimera
. All MD simulations were performed using the
[19]
GROMACS 4.5.6 version
with AMBER99SB-ILDN force field [20], as
employed previously [8b, 21]. Long-range electrostatics were handled using
Particle Mesh Ewald method [22]. Ligand topology and parameters were
generated by applying AM1-BCC charges and Generalised Amber Force
Field [23] using a python script Acpype [24] that uses Antechamber module
[25]
of AMBER
. The protein-ligand complexes were initially energy
minimised using the steepest descent method, followed by position
restrained minimisation and finally the 100 ns production run. Hydrogen
bond analysis was performed using the g_hbond utility available with the
GROMACS package, and the occupancy analysis was performed using
the python script readHBmap, written by R. O. S. Soares. Visualisation of
MD trajectories was carried out in VMD [26]. The ligand binding free
energy was estimated using molecular mechanics energies with the
Methyl
2-O-methoxymethyl-4,6-O-benzilidene-β-D-galactopyranoside
Poisson-Boltzmann and surface area continuum solvation method
(4): Compound 3 was dissolved in MeOH and cooled to 0 ᵒC before
addition of sodium metal previously suspended in hexane. The reaction
was left at room temperature for 1.5 hours, then carefully acidified to pH
5 using 1 M HCl. Salts were removed by water washing, and the product
was extracted with EtOAc, then solvent removed in vacuo to give 4 (90%
yield). 1H NMR (CD3OD, 400 MHz): δ 3.35 (3H, s, MOM), 3.47 (1H, s),
3.48 (3H, s, OCH3), 3.56 (1H, dd, J= 7.6, 9.6 Hz), 3.62 (1H, dd, J= 3.6,
9.6 Hz), 4.06 (1H, d, J=1.6, 12.4 Hz), 4.13 (2H, m, J=1.6 Hz), 4.24 (1H, d,
J=7.6 Hz), 4.66 (1H, d, J=6.4 Hz), 4.76 (2H, s, MOM), 5.53 (1H, s, CHPh),
7.33 (3H, m), 7.33 (3H, m), 7.49 (2H, m). 13C NMR (CD3OD, 100 MHz): δ
= 54.7, 55.8, 66.5, 68.7, 71.8, 75.6, 76.3, 96.7, 101.1, 104.6, 126.0,
127.0, 128.5, 138.3. MS (ESI): m/z calculated for C16H22NaO7 [M+Na]+
349.1, found 349.1.
[27]
(MMPBSA.py)
implemented in Amber package [28]. A set of 100
frames periodically extracted from the trajectory file at an interval of 1 ns
were subjected to MMPBSA analysis to obtain the ligand binding free
energies.
Chemical Synthesis
General procedure: Thin Layer Chromatography (TLC) on pre-coated
aluminium-backed silica plates (60 F254; Merck) were used to assess
reaction progression by visualisation after charring in 4% sulphuric acid
in ethanol. Reaction products were purified using flash chromatography
silica gel 60. 1H and 13C NMR spectra were recorded at 298 K using an
Avance Bruker Biospin spectrometer (400 and 100 MHz, respectively;
Bruker Biospin) spectrometer. Two-dimensional COSY (1H to 1H
correlation), HSQC (1H to 13C correlation) and HMBC (1H to 13C long
range correlation) NMR experiments were used to assist in assigning
relevant peaks for 1H and 13C NMR spectra. Electrospray ionisation low-
resolution mass spectrometry was performed using a Bruker Daltronics
Esquire 3000 Ion-Trap MS.
Methyl 3-O-[1-carboxyethyl]-β-D-galactopyranoside (6): The propionic
acid side chain was installed onto 4 using previously reported conditions
[13]. Compound 4 was dissolved in anhydrous 1,4-dioxane under argon
and cooled to 0ᵒC before addition of NaH. 2-Chloropropionic acid was
slowly added at 0 ᵒC, then the reaction was stirred at 50 ᵒC for 36 hours
to yield the racemic mixture of novel ligand 5 (60% yield). 1H NMR
(CDCl3, 400 MHz): δ = 1.64 (3H, d, J=6.8 Hz, CH3CH), 3.36 (3H, s,
OCH3), 3.39 (1H, m), 3.48 (3H, s, OCH3), 3.61 (1H, dd, J=3.2, 9.6 Hz),
3.77 (1H, dd, J=8.0, 10.0 Hz), 4.16 (2H, m, J=1.2, 12.4 Hz, H6), 4.38 (3H,
m, J=8.0 Hz), 4.51 (1H, d, J=3.2 Hz), 4.78 (1H, d, J=6.4 Hz), 5.62 (1H, s,
CHPh), 7.25-7.34 (3H, m), 7.40-7.49 (2H, m). 13C NMR (CD3OD, 100
MHz): δ = 18.1, 19.2, 56.5, 56.0, 56.9, 57.0, 66.3, 65.9, 69.0, 69.1,72.5,
72.8, 73.6, 74.0, 75.2, 75.3, 79.5, 81.3, 97.5, 98.2, 101.1, 101.7, 103.7,
103.8, 126.3, 126.6, 128.2, 128.4, 129.1, 129.5, 136.8, 137.5, 173.6,
[10]
Methyl 4,6-O-benzilidene-β-D-galactopyranoside (1)
: Methyl-β-D-
galactopyranoside (1 g, 5.1 mmol) was dried in vacuo overnight before
being dissolved in anhydrous acetonitrile (CH3CN) (10 mL) under argon.
A catalytic amount of camphor sulfonic acid (CSA) (52 mg, 0.22 mmol)
was added followed by dropwise addition of benzaldehyde dimethyl
acetal (1.5 mL, 14.8 mmol) added dropwise. The reaction was heated to
60 ᵒC for about 3 hours. The reaction was quenched with triethylamine
(Et3N), then purified via flash chromatography using 40:1
6
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10.1002/cmdc.201800224
ChemMedChem
FULL PAPER
[33]
174.0. MS (ESI): m/z calculated for C19H26NaO9 [M+Na]+ 421.2, found
421.3.
implemented in CCP4
was used for molecular replacement with the
apo galectin-8N (PDB ID: 3AP5 [8a]) structure used as the search model.
The chemical information file for 6 was generated using the PRODRG
[34]
server
.
Refinement was carried out using REFMAC5 [35], model
The deprotection of 5 was carried out in MeOH and concentrated HCl at
room temperature. The enantiomeric mixture of novel ligand 6 was
purified using reversed-phase chromatography (C18, 4:1 water:MeOH).
1H NMR (CD3OD, 400 MHz): δ = 1.23 (3H, d, J=8.0 Hz), 3.33 (1H, dd,
J=3.2, 9.6 Hz), 3.41 (3H, s, OCH3), 3.40-3.66 (4H, m), 3.92 (1H, m), 3.98
(1H, q, J=8.0 Hz, CHCH3), 4.18 (1H, dd, J=6.0, 8.0 Hz). 13C NMR
(CD3OD, 100 MHz): δ = 17.6, 55.8, 61.0, 67.2, 70.7, 74.9, 75.2, 82.1,
building, and visualisation done in COOT [36]. Final model validation
performed using MolProbity [37]. Molecular graphics and electron density
illustrations for figures were performed using the UCSF Chimera package
[18, 38]
.
+
104.5. MS (ESI): m/z calculated for C10H18KO8 [M+K] 305.4, found
306.1.
Acknowledgements
H.B gratefully acknowledges the financial support from the
Cancer Council Queensland (ID1080845).
Protein expression and purification
The galectin-8N protein was expressed in an untagged form as described
previously [8b]. Briefly, the bacterial culture was induced with IPTG for 4
hours at room temperature and purified using affinity chromatography on
a lactosyl-Sepharose column at 4 °C. The purity of the expressed protein
was assessed using SDS-PAGE and was used directly for binding
assays and crystallisation.
Accession code: Protein Data Bank: Atomic coordinates and
structure factors have been deposited with PDB accession code
5VWG for the galectin-8N-methyl 3-O-[1-carboxyethyl]-β-D-
galactopyranoside (6) complex.
Keywords: Structure-based ligand design • carbohydrate •
galectin-8N terminal domain • galectin-8C terminal domain • X-
ray crystal structure
Galectin-8C was expressed with 10x His-tag by using pET-19b vector in
the E. coli BL21(DE3) strain. The E. coli. cells were grown in Luria-
Bertani (LB) medium supplemented with 100 μg/ml Ampicillin to reach an
OD600 of 0.8. The culture was induced with 0.25mM IPTG and incubated
at 16°C for 18 hrs to reach the optimum yield. To extract the cytoplasmic
soluble protein, cells were lysed using sonication and centrifuged to
harvest the supernatant. Galectin-8C was purified using affinity
chromatography on Ni-NTA column equilibrated with PBS, pH 7.9,
containing 10mM imidazole. Protein was then eluted using 370mM
imidazole and dialyzed against Tris buffer with 2.5 mM b-
mercaptoethanol (BME). The purified protein was concentrated using
Amicon Ultra centrifugal filter units (Millipore, Billerica, MA, USA) with a
3kDa molecular weight cut-off (MWCO). The purity of the expressed
protein and obtained protein fractions were assessed by SDS-PAGE.
Protein concentration was determined using UV spectroscopy at 280 nm.
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8
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Entry for the Table of Contents
Exploiting both evolutionarily conserved and unique binding-site amino acids in ligand design: Targeting human galectin-8,
we have undertaken in silico structure-based design and chemical synthesis to generate a novel efficient ligand, and validated its
binding by ITC and X-ray crystallography.
9
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