Inhibitors against Fungal Cell Wall Remodeling Enzymes

Article abstract of DOI:10.1002/cmdc.201700720

Fungal β-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inh

Full text of DOI:10.1002/cmdc.201700720

Accepted Article
Title: Inhibitors against Fungal Cell Wall-remodelling Enzymes
Authors: Pedro Merino, Ignacio Delso, Jessika Valero-Gonzalez,
Fernando Gomollon-Bel, Jorge Castro-Lopez, Wenxia Fang,
Iva Navratilova, Daan M. F. van Aalten, Tomas Tejero, and
Ramon Hurtado-Guerrero
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201700720
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Inhibitors against Fungal Cell Wall-remodelling Enzymes.
Ignacio Delso,*[a] Jessika Valero-Gonzalez,[b] Fernando Gomollón-Bel,[a] Jorge Castro-López,[b] Wenxia
Fang,[c] Iva Navratilova,[c] Daan M.F. van Aalten,[c] Tomás Tejero,[a] Pedro Merino,*[b] and Ramon
Hurtado-Guerrero*[b,d]
Abstract: Fungal -1,3-glucan glucanosyltransferases are glucan-
remodeling enzymes that play important roles in cell wall integrity, and
are essential for the viability of pathogenic fungi/yeasts. As such, they
are considered possible drug targets, although inhibitors of this class
essential for viability of Schizosaccharomyces pombe.[6] In fungi,
the gene encoding the TG Gel4 in A. fumigatus is also essential
for viability.[4] This family of TGs is grouped into 3 subfamilies
based on the presence or absence of CBM43, a carbohydrate
binding module most commonly associated with β(1-3)glucan
binding activity and a serine/threonine-rich region that contains O-
glycosylation putative sites.[4,7] We previously reported the crystal
structure of ScGas2 in complex with laminarioligossacharides[7]
and recently trapped a covalent intermediate on the pathway to
transglycoslation (Figure 1).[8] From these structures, we defined
the different sites for the sugar moieties bound in the donor and
acceptor sites, and we also verified the role of the proposed acid-
base Glu176 and the nucleophile Glu275.
of enzymes have not yet been reported. Here, we report
a
multidisciplinary approach, based on a structure-guided design using
a highly conserved transglycosylase from Sacharomyces cerevisiae,
that leads to carbohydrate derivatives with high affinity for Aspergillus
fumigatus Gel4. We demonstrate by X-ray crystallo-graphy that the
compounds bind in the active site of Gas2/Gel4 and interact with the
catalytic machinery. The topological analysis of non-covalent
interactions demonstrates that the combination of a triazole with
positively charged aromatic moieties are important for optimal
interactions with Gas2/Gel4 through unusual pyridinium cation-π and
face-to-face π-π interactions. The lead compound is capable of
inhibiting AfGel4 with an IC50 value of 42 M.
The fungal cell wall is essential for the integrity of the cell,
providing strength, shape and protection against environmental
insults.[1] However, rather than being a rigid structure it has
inherent plasticity during cell growth and adaptation to the
environment.[2] Cell wall remodelling enzymes and in particular
the large family of -1,3-glucan glucanosyltransferases or
transglycosylases (TGs) contribute to this plasticity.[3] These
enzymes remodel the polysaccharide -1,3-glucan, which is the
major component of the fungal cell wall. They cleave the β-1,3
bond of β-1,3-glucan oligosaccharides with at least 10 glucose
units and transfer the newly formed reducing end (>5 glucose
units) to the nonreducing end of another β-1,3-glucan
oligosaccharide, resulting in the elongation of the β-1,3-glucan.[4]
These TGs have been found in all fungal species investigated to
date and are named differently depending on the fungi or yeasts
in which they are found (e.g. Gelp, Gasp and Phrp in A. fumigatus,
S. cerevisiae and C. albicans, respectively).[5] In yeasts, these
TGs have important roles in spore and hyphal wall assembly, and
maintaining cell wall integrity during vegetative growth, and are
Figure 1. View of the active site of the covalent intermediate of E176Q ScGas2
with laminaritetraose (donor site) in complex with laminaripentaose (acceptor
site; see PDB ID: 5FIH [ref]). The carbons of the oligosaccharides are shown as
yellow sticks. Compound 1 (carbons shown as cyan sticks) docked in ScGas2
active site is superposed with the above structure. Residues as grey sticks are
involved in interactions with the sugar moieties and compound 1. Note that
laminaritetraose is covalently bound to E275.
[a] Dr. I. Delso, Dr. F. Gomollón-Bel and Prof. T. Tejero
Instituto de Síntesis Química y Catálisis Homogénea (ISQCH).
Universidad de Zaragoza. CSIC. E-50009 Zaragoza. Aragón, Spain.
E-mail: idelso@unizar.es
[b] Dr. J. Valero-González, J. Castro-López, Dr. R. Hurtado-Guerrero and
Prof. P. Merino
Institute of Biocomputation and Physics of Complex Systems (BIFI),
University of Zaragoza. BIFI-IQFR (CSIC) Joint Unit, Campus Rio
Ebro. E-mail: rhurtado@bifi.es; E-mail: pmerino@unizar.es
[c] Dr. W. Fang, Dr. I. Navratilova, and Prof. D.M.F. van Aalten; Centre for
Gene Regulation and Expression, School of Life Sciences, University
of Dundee, Dundee DD1 5EH, Scotland
Although these enzymes are potential antifungal drug targets,
there are currently no potent inhibitors of this family of enzymes.
We previously synthetized[9] and characterised compound 1, a
laminaritriose derivative bound to a phenyl moiety through a
flexible linker, which showed a poor affinity (Kd = 800 ± 100 µM)
towards Gas2 (Figure 1). We used ScGas2 for our studies here
because it is a homologue of AfGel4, a proposed antifungal drug
target, and its crystal structure has been reported.[7] ScGas2 is
[d] Fundación ARAID, 50018, Zaragoza, Spain.
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10.1002/cmdc.201700720
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similar to AfGel4 at the sequence level (ca. 50% sequence identity
trisaccharide unit. Monosaccharides 3-4 and disaccharide 5
showed high Kd values, reinforcing our finding that three units of
trisaccharide are required as a minimum recognition scaffold.
in the catalytic domains, and 100% if considering all the key
residues recognising carbohydrates from -3 to +1; see Figure
S1).[7] The rationale for making compound 1 was to occupy the
donor site with the sugar moieties by interacting with the
conserved Pro136 and Tyr107, and reach the acceptor site
establishing CH-pi interactions between the phenyl moiety and
the conserved Tyr244, as exemplified by a docked model of
compound 1 in the ScGas2 active site (Figure 1). In this model,
the trisaccharide moiety occupies the -2 to -4 subsites and two
conserved tyrosine residues (Tyr107 and Tyr244) interact with the
sugar unit occupying position -1 (carbohydrate-π interactions)[10]
and the phenyl group of at the ligand tail (CH-π interactions).[11]
Taking advantage of the high structural homology and the
complete conservation of the active site among AfGel4 and
ScGas2, we reasoned that the introduction of an additional
aromatic moiety interacting an additional conserved tyrosine
(Tyr307, Figure 1) could result in an enhanced binding for both
AfGel4 and ScGas2. Simultaneously, it should also be possible to
explore different aromatic residues at the ligand tail to enhance
interactions with Tyr244.
OH
OH
OH
O
O
O
HO
O
HO
O
HO
HO
O
OH
OH
OH
N3
Cu(OAc)2 (2%), Cu(0)
H2O / MeOH
OH
OH
OH
N
N
O
O
O
HO
O
HO
O
N
HO
HO
O
OH
OH
OH
2
O
O
n = 1; R =
n = 2; R =
n = 1; R =
3
4
OH
O
HO
O
N3
O
H
OH
n
5
6
Cu(OAc)2 (2%), Cu(0)
H2O / MeOH
R
O
O
O
7
In this study, we explore the use of a triazole scaffold to reach
Tyr307 taking advantage of two amenable and efficient synthetic
strategies based on click chemistry.[12] We demonstrate the
strength of this strategy by synthesizing eleven different
candidates and determining their dissociation constants (Kds)
towards ScGas2 and AfGel4. The results demonstrate that the
laminaritriose moiety directly bound to a triazole together with
positively charged aromatic moieties at ligand tail are important
for an optimal interaction with these enzymes. The observed
results are confirmed by solving the crystal structures of ScGas2
O
O
OH
n = 3; R =
N
S
N
N
N
R
O
O2N
8
9
10
HO
O
H
OH
N
N
n
11
12
Scheme 1. Chemical synthesis of ligands through typical click-chemistry.
Table 1. Dissociation constants of compounds 1-12 for ScGas2 and AfGel4.
in complex with
7 out of the 11 compounds by X-ray
crystallography studies in combination with computational NCI
analyses. These data also show that hydrophobic interactions,
particularly π-π and azinium-π (cation-π) interactions, between
Tyr244 and the aromatic moieties at the ligand tail, as well as
between the triazole moiety and Tyr307, are the determinants of
the increased affinity for ScGas2.
entry
1
Compound
Kd (ScGas2) (M)
800 ± 100
17 ± 0.1
Kd (AfGel4) (M)
nd[b]
1[a]
2
2
nd
3
3
171 ± 1
nd
Introduction of the triazole ring into the linker was achieved
through the well-known CuAAC reaction applied to carbohydrate
chemistry (Scheme 1).[13] Two alternative coupling strategies for
4
4
72 ± 1
nd
5
5
68 ± 1
nd
preparing
the
triazole
ring
were
explored,
i.e.:
laminarioligosaccharide alkyne with a properly substituted azide
and laminarioligosaccharide azides[|4] with properly substituted
alkynes. Compound 2, an analogue of compound 1 containing a
triazole ring in the linker, was prepared following the first strategy.
However, docking studies with 2 (Glide, see SI) showed that the
triazole ring did not reach the target Tyr307. In fact, with ScGas2
we obtained a value of Kd = 17.0 ± 0.1 µM for 2 (Table 1, entry 2),
higher than the Kd for the equivalent compound 6, which differed
in the position of the triazole (Kd = 6.2 ± 0.1 µM; see below for its
synthesis).
6
6
6.2 ± 0.1
6.00 ± 0.06
10.0 ± 0.1
nd
7
7
13.00 ± 0.01
9.80 ± 0.07
4.09 ± 0.04
8.06 ± 0.07
4.60 ± 0.03
3.10 ± 0.02
8
8
9
9
3.60 ± 0.03
5.30 ± 0.06
2.50 ± 0.02
1.50 ± 0.01
10
11
12
10
11
12
Docking studies suggested that a triazole positioned closer to
the sugar units as well as electron-poor aromatic residues at the
ligand tail would increase the affinity of the ligand (See SI).
Consequently, we prepared compounds 3-12 following the
strategy illustrated in Scheme 1. In order to evaluate the effect in
binding of the carbohydrate units we also prepared mono- (3-4)
and disaccharides (5) in addition to potential ligands bearing a
[a] = determined by tryptophan fluorescence spectroscopy; [b] nd = not
determined. Laminaritriose showed too weak affinity to be measured.
Aromatic residues in compounds 6 and 7 lowered the Kd
values for ScGas2, similar values being observed for AfGel4 in
line with a high identity between enzymes. Increasing of the size
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ring from phenyl (6) to naphthyl (7) did not improve binding affinity.
E). One conformation corresponds to the neutral ligand folded on
itself and stabilized by intramolecular π-π interactions with the
thiazole ring and H-bonding with a water molecule, which is also
interacting with E176 and E275. Notably, the other conformation
corresponds to a protonated pyridine ring, which adopts a parallel
orientation with respect to Tyr244. This favours a transfer of
charge between the Tyr244 electron-rich aromatic ring and the
electron-poor pyridinium ring through pyridinium−π (cation−π)
interaction, which has been shown to be stronger than the typical
face-to-face π−π interactions,[16] rarely observed in protein
structures where most of the orientations are T-shaped (also
named as edge-to-face).[17]
Employing an electron withdrawing substituent on the aromatic
ring we observed an intermediate value and with the thienyl-
derivative 9 (Table 1, entry 9), we obtained one of the lowest Kd
values observed for both ScGas2 and AfGel4. In searching for
stronger π-π interactions by introducing π-deficient aromatic rings
which can interact in a more efficient way[15] with the electron rich
aromatic ring of tyrosine, we moved to the pyridine derivative 10
which, at physiological pKa is amenable to be protonated to some
extent as demonstrated by NMR experiments (see SI). Moderate
Kd values were obtained for both enzymes (Table 1, entry 10).
Using positively charged pyridinium moiety as in 11 (Table 1,
entry 11) reduced the Kd, particularly for AfGel4, which was further
enhanced when we moved from pyridinium 11 to quinolinium
derivative 12 (Table 1, entry 12). Compound 12 showed Kd = 3.10
± 0.02 and 1.50 ± 0.01 µM, against ScGas2 and AfGel4,
respectively, being the lowest found among all the prepared
glycomimetics. Figure 2 illustrates determination of the Kd for
compound 12 towards ScGas2 by SPR. In addition, we could not
obtain a Kd for laminaritriose due to its low affinity. This result
points to the introduced groups (triazole ring and aromatic
residues) as the responsible of the tight binding of the designed
glycomimetics to both enzymes.
Figure 2. Surface plasmon resonance (SPR) measurements for compound 12
towards ScGas2. The various concentrations of 12 show the binding reaction
and the calculated dissociation constant is 3.1 ± 0.02 μM. Plain brown lines
show the fitted curve of non-linear regression.
To elucidate the binding mode of the designed glycomimetics we
solved the crystal structures of compounds 6-12 in complex with
ScGas2 at high resolution, ranging from 1.4 to 2.15 Å (Figure 4,
ESI). In all cases, the trisaccharide unit occupied the donor site in
the -2, -3 and -4 positions. The triazole ring was observed to form
π-π stacking interactions with Y307. The observed interactions of
the aromatic residue located at the ligand tail were different
depending on the ring type. Thus, whereas ligands 6-9 adopt a T-
shape (edge-to-face) orientation of the aromatic residues with
respect to Tyr244, positively charged residues in 11 and 12 adopt
a parallel orientation. The case of pyridine derivative 10 deserves
a more detailed discussion.
In addition, for compounds 6, 8 and 9, another ligand molecule is
observed at the acceptor site, likely due to the less optimal
interactions between the radical at the tail position with Y244 (see
Figure S2). The observed T-shape orientation observed for
compounds 6-9 corresponds to a typical CH-π interaction, as that
observed in the docked structure of 1. In the case of compound
10, two conformations were found in the X-ray structure (Figure 3,
Figure 3. X-ray structures of ScGas2 in complex with 6 (A), 7 (B), 8 (C), 9 (D),
10 (E), 11 (F) and 12 (G). The unbiased (i.e. before inclusion of any ligand
model) |Fo |- |Fc |, calc electron density maps are shown at 2.2 .
To evaluate the conformational preferences, we carried out a
molecular dynamics (MD) study of ScGas2 in complex with
compound 10 in folded/unfolded and protonated/neutral states
(Figure 4). MD calculations showed that both protonated and
neutral folded derivatives become unfolded after 0.6 and 2.5 ns,
respectively. This result suggests that the unfolded conformation
is the most representative. The results observed with compound
10 indicate that the presence of a positively charged π-deficient
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aromatic residue induces a change of the relative orientation
interactions with positively charged aromatic residues favours a
parallel orientation of the aromatic rings, which enhances the
strength of the resulting azinium-π (cation-π) interactions.
Understanding the ultimate conditions to design a good ligand for
ScGas2 we obtained a first low micromolar inhibitor for AfGel4
demonstrating the validity of the approach. These studies will
open the door to the design of more potent inhibitors of AfGel4
that could be useful as a platform to discover antifungal
therapeutic agents.
between the aromatic residue and Tyr244. Indeed, when the
positive charge is permanent (at physiological pH) as in the case
of aziniums 11 and 12, only unfolded conformations with a parallel
orientation to Tyr244 are found in the X-ray structure. The parallel
orientation allows more efficient π,π-interactions with Tyr244 in
comparison with CH-π interactions observed in compounds 6-9.
The existence of these interactions was confirmed through a
topological NCI analysis[18] that showed the expected surfaces for
such a sort of non-covalent interactions (Figure 5).
Acknowledgements
This work was supported by Spanish MINECO Contracts
(CTQ2016-76155-R to P.M., and BFU2016-75633-P to R.H-G.),
and an MRC Programme Grant (M004139) to D.M.F.v.A. We also
acknowledge the Government of Aragón (Spain) (Bioorganic
Chemistry group E-10 and Protein Targets group B-89) for
financial support. We acknowledge the Institute of
Biocomputation and Physics of Complex Systems (BIFI) at the
University of Zaragoza (Spain) for computer time at clusters
Terminus and Memento. We thank synchrotron radiation sources
DLS (Oxford), and in particular beamline I02 (experiment number
MX10121-11), and ALBA (Barcelona), and in particular XALOC
beamline. European Commission is gratefully acknowledged
(BioStruct-X
grant
agreement
N°283570
and
BIOSTRUCTX_5186).
Figure 4. Molecular dynamics of 10 showing unfolding after 2.5 ns and its
maintenance up to 30 ns (extension to 100 ns do not show any change). Starting
structures: red trace: protonated folded; green trace: neutral folded; black trace:
protonated unfolded; blue trace: neutral unfolded
Keywords: transglycosylases • Aspergillus fumigatus •
oligosaccharides • glycomimetics • carbohydrates
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an IC50 value of 42.0 M (See SI), validating our approach of using
ScGas2 as a model for the design of glycomimetics against
AfGel4.
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ScGas2 as a model, we have obtained quinolinium derivative 12
showing a Kd of 1.50 ± 0.01 µM and inhibitory properties against
AfGel4 (IC50 = 42 µM). During the design, π,π-interactions with
Tyr307 and Tyr244 were discovered to be the driving force for
achieving optimal binding. In particular, in the case of Tyr244,
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COMMUNICATION
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[19] The biological evaluation of 12 towards AfGel4 was carried out through
a
competition assay using
a
mixture of G14- and G15-
laminooligosaccharides, where their disappearance was quantified by
RP-UPLC.
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Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Structure-guided design using a
transglycosylase from
Saccharomyces cerevisiae as a
model led to a micromolar inhibitor
of pathogenic Aspergillus
fumigatus
I. Delso,* J. Valero-Gonzalez, F.
Gomollón-Bel, J. Castro-López, W.
Fang, I. Navratilova, D. M.F. van
Aalten, T. Tejero, P. Merino,* and R.
Hurtado-Guerrero*
Page No. – Page No.
Inhibitors against Fungal Cell
Wall-remodelling Enzymes .
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