Inhibition of influenza virus infection by multivalent pentacyclic triterpene-functionalized per-O-methylated cyclodextrin conjugates

Article abstract of DOI:10.1016/j.ejmech.2017.03.087

Multivalent ligands that exhibit high binding affinity to influenza hemagglutinin (HA) trimer can block the interaction of HA with its sialic acid receptor. In this study, a series of multivalent pentacyclic triterpene-functionalized per-O-methylated cyclodextrin (CD) derivatives were designed and synthesized using 1, 3-dipolar cycloaddition click reaction. A cell-based assay showed that three compounds (25, 28 and 31) exhibited strong inhibitory activity against influenza A/WSN/33 (H1N1) virus. Compound 28 showed the most potent anti-influenza activity with IC₅₀ of 4.7?μM. The time-of-addition assay indicated that compound 28 inhibited the entry of influenza virus into host cell. Further hemagglutination inhibition (HI) and surface plasmon resonance (SPR) assays indicated that compound 28 tightly bound to influenza HA protein with a dissociation constant (K_D) of 4.0?μM. Our results demonstrated a strategy of using per-O-methylated β-CD as a scaffold for designing multivalent compounds to disrupt influenza HA protein-host receptor protein interaction and thus block influenza virus entry into host cells.

Full text of DOI:10.1016/j.ejmech.2017.03.087

European Journal of Medicinal Chemistry 134 (2017) 133e139
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Inhibition of influenza virus infection by multivalent pentacyclic
triterpene-functionalized per-O-methylated cyclodextrin conjugates
,
,
Zhenyu Tian a 1, Longlong Si a 1, Kun Meng a, Xiaoshu Zhou a, Yongmin Zhang b,
Demin Zhou a, Sulong Xiao a
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
,
*
b
ꢀ
ꢀ
Sorbonne Universites, UPMC Univ Paris 06, Institut Parisien de Chimie Moleculaire, CNRS UMR 8232, 4 place Jussieu, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Multivalent ligands that exhibit high binding affinity to influenza hemagglutinin (HA) trimer can block
the interaction of HA with its sialic acid receptor. In this study, a series of multivalent pentacyclic
triterpene-functionalized per-O-methylated cyclodextrin (CD) derivatives were designed and synthe-
sized using 1, 3-dipolar cycloaddition click reaction. A cell-based assay showed that three compounds
(25, 28 and 31) exhibited strong inhibitory activity against influenza A/WSN/33 (H1N1) virus. Compound
Received 17 January 2017
Received in revised form
22 February 2017
Accepted 31 March 2017
Available online 2 April 2017
28 showed the most potent anti-influenza activity with IC50 of 4.7
mM. The time-of-addition assay
Dedicated to Professor Lihe Zhang on the
Occasion of His 80th Birthday.
indicated that compound 28 inhibited the entry of influenza virus into host cell. Further hemaggluti-
nation inhibition (HI) and surface plasmon resonance (SPR) assays indicated that compound 28 tightly
bound to influenza HA protein with a dissociation constant (KD) of 4.0
mM. Our results demonstrated a
Keywords:
strategy of using per-O-methylated -CD as a scaffold for designing multivalent compounds to disrupt
b
Per-O-methylated cyclodextrin
Pentacyclic triterpene
Influenza virus
influenza HA protein-host receptor protein interaction and thus block influenza virus entry into host
cells.
© 2017 Elsevier Masson SAS. All rights reserved.
Hemagglutinin
Multivalent effect
1. Introduction
protein to its cellular receptor, sialic acid (SA)-linked glycoproteins,
followed by fusion of the viral and endosomal membranes.
Influenza A viruses are enveloped viruses belonging to the
family Orthomyxoviridae. They can cause both annual epidemics
and occasional pandemics and remain an enormous clinical and
public health challenge worldwide [1]. Based on the antigenic
major surface glycoproteins hemagglutinin (HA; 18 subtypes) and
neuraminidase (NA; 11 subtypes), they are divided into different
subtypes [2]. Currently available options to fight against the res-
piratory system attack by influenza A viruses include both vaccines
and antiviral agents. However, the three types of available vaccines,
inactivated influenza vaccine, live attenuated influenza vaccine, and
recombinant influenza vaccine, have moderate efficacy that varies
seasonally due to constant viral evolution [3]. The biggest challenge
in the development of anti-influenza therapeutics is the rapid
emergence of drug-resistant strains due to the high mutation rate
[4e7]. Influenza A virus infection begins with the binding of HA
Consequently, effective blocking of the interaction between HA and
SA could lead to suppression of viral infection [8,9].
Structurally, HA is composed of a head domain (HA1), which
contains the receptor-binding site, and a stem domain (HA2/HA1)
[10,11]. It is synthesized as a single polypeptide precursor protein
(HA0). Three copies of HA assemble into a noncovalent trimer
which mediates influenza A virus infection by attaching to SA re-
ceptors on the cell surface to facilitate virion internalization [10,12].
Once inside the acidic endosome, a conformational change in the
trimeric structure occurs, exposing a fusion peptide and allowing
endosomal escape of the viral RNA. Low-angle neuron scattering
studies have shown that the surface glycoproteins, predominantly
HA trimers, constitute 40e60% of the virus mass [13,14]. The high
density of HA on the surface of influenza virions results in specific
high-avidity binding of the virus to the mucosal membranes [15].
This process provides important clues for the design of efficient
multivalent ligands to block the viral protein-host receptor in-
teractions [16].
* Corresponding author.
Cyclodextrins (CDs) are cyclic oligosaccharides, containing a
relatively hydrophobic central cavity and hydrophilic outer surface
E-mail address: slxiao@bjmu.edu.cn (S. Xiao).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2017.03.087
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

134
Z. Tian et al. / European Journal of Medicinal Chemistry 134 (2017) 133e139
[17]. They possess unique ability to act as molecular containers by
entrapping guest molecules in their internal cavity [18,19]. The
unique steric accessibility and acidity of the three types of hy-
droxyls in CDs have been taken into account to conceive efficient
position-selective and face-selective chemical functionalization
methodologies [20]. Over the past decades, a variety of multivalent
conjugates based on CD scaffold involving heptylmannoside-
functionalized glycoconjugate [21], mannose-functionalized
2. Results and discussion
2.1. Chemistry
Synthesis of the multivalent betulinic acid (BA)-CD conjugates
has been performed as shown in Scheme 1. We selected
-CD as the central building blocks to describe the structurally
diverse collection of multivalent conjugates. At first, compounds
4e6 were synthesized from the commercially available -, - and
a-, b- and
g
amphiphilic
b-CDs [22], and other biorecognizable sugar ligand-
a
b
g-
functionalized CDs [23], have been prepared and shown to be
effective modulators of carbohydrate-protein interactions. In our
previously study, a series of pentacyclic triterpenes were grafted
onto the primary face of CD to explore the interactions between
multiple pentacyclic triterpenes and HA protein; a dramatic
multivalent effect for their anti-influenza virus entry activity has
been found [24].
In spite of the progress, the design of multivalent ligands
with optimized biological properties still remains a challenge
considering the complexity of the multivalent ligand-receptor
interactions [25]. Recently, per-O-methylated CDs and their de-
rivatives have attracted considerable attention due to their
improved solubility in water and organic solvents [26,27]. In
addition, they show strong binding ability to anionic tetraar-
ylporphyrin [28] and can be used to functionalize the bovine
serum albumin (BSA) surface for inducing self-assembly [29].
However, to our knowledge, no multivalent compound has been
designed based on per-O-methylated CD. As a natural step in our
efforts towards the development of novel antiviral inhibitors
[24,30], we prepared multivalent pentacyclic triterpene-
CD by primary face-selective functionalization according to the
procedure described by Ashton et al. [31] This was followed by
nucleophilic substitution with sodium azide in N,N-dime-
thylformamide to provide the intermediate per-(6-azide-6-deoxy)-
CDs (7e9) in quantitative yield, which was used without further
purification in the next step. Methylation of crudes 7e9 with CH3I
in the presence of NaH afforded the per-(6-azide-6-deoxy-2,3-di-O-
methyl)-CDs (10e12) in 65e77% yield. The synthesis of alkynyl-
functionalized BA derivative 17 was performed from betulin 13
with 49% yields in four steps according to the reported method [32].
In the last step, coupling of 17 with azide functionalized com-
pounds 10e12 via click reaction in THF/H2O (1:1) in the presence of
a catalytic amount of copper sulfate and L-sodium ascorbate yiel-
ded the multivalent BA-functionalized per-O-methylated CD con-
jugates 18e20, respectively.
Similarly, the synthesis of alkynyl-functionalized pentacyclic
triterpenes derivatives 21e23 were performed from commercially
available oleanolic acid (OA), echinocystic acid (EA) and ursolic acid
(UA) with high yields in two steps. Subsequent conjugation with
10e12 in a similar manner as described in Scheme 1 afforded
conjugates 24e32 in yields ranging from 43 to 65% (Scheme 2).
The structures of pentacyclic triterpene-functionalized per-O-
methylated CD conjugates 18e20 and 24e32 were characterized by
functionalized per-(2,3-di-O-methyl)-
a-, b- or g-CD conjugates
and evaluated their in vitro anti-influenza virus activities.
Scheme 1. Reagents and conditions: (a) Ph3P, I2, DMF, 80 ꢀC, overnight; (b) NaN3, H2O, 60 ꢀC, 12 h; (c) CH3I, NaH, DMF; (d) H2CrO4, acetone, 0 ꢀC to rt, 18 h; (e) NaBH4, THF, rt, 2.5 h; (f)
TBTU, DIPEA, THF; (g) 2-propargylamine, K2CO3, DMF, 1 h; (h) CuSO4, Na-L-ascorbate, THF-H2O (1:1, V/V), 100 ꢀC, microwave.

Z. Tian et al. / European Journal of Medicinal Chemistry 134 (2017) 133e139
135
Scheme 2. Reagents and conditions: (a) CuSO4, Na-L-ascorbate, THF-H2O (1:1, V/V), 100 ꢀC, microwave.
Fig. 1. A portion of the 400 MHz HSQC (CDCl3, 25 ꢀC) spectrum of 18, with the 1D 1H and 13C NMR spectra along the top and the side, respectively.

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Z. Tian et al. / European Journal of Medicinal Chemistry 134 (2017) 133e139
Table 1
one and two dimensional NMR and MALDI-TOF spectroscopy. Fig. 1
represents the HSQC spectrum of compound 18 as an example. In
the aromatic region, the signal at 7.53 ppm was assigned to triazole-
CH according to the 1H-1H and 1H-13C correlation spectra. The
assignment of NH proton at 6.82 ppm was confirmed as there is no
correlation for NH by HSQC spectral analysis. With a C6-symmetry
in the molecule, compound 18 showed only one set of CD-H1 signal
appearing at 5.31 ppm and one CD-C1 signal appearing at
99.09 ppm. Similar observation was also made for other proton and
carbon signals due to the C6-symmetry. The MALDI-TOF MS of
compound 18 showed a sodium adduct ion at m/z 4273.9, which
further confirmed it was a hexavalent conjugate.
Inhibitory activities of multivalent conjugates 18e20 and 24e32 against infection of
MDCK cells by influenza A/WSN/33 virus.
Compound
Oseltamivirc >200
18
19
20
24
25
26
CC50 (m (m (m (m
M)a IC50 M)b Compound CC50 M)a IC50 M)b
16.5 [24]
>100
>100
>100
>100
>100
>100
14.5 1.74 27
22.8 1.48 28
23.0 1.59 29
48.0 5.04 30
>100
52.4 7.86
4.7 0.52
30.9 4.33
40.3 3.63
6.5 1.24
>100
>100
>100
>100
>100
>100
9.9 0.79
31
32
>100
a
50% cytotoxicity concentration.
Concentration inhibiting viral replication by 50%. The values are means of at
b
least three independent determinations; the corresponding standard deviations are
noted.
Fig. 2 presents the characteristic portions (80e180 ppm) of the
13C NMR spectra (in CDCl3) of conjugates 18, 27, and 30. The signals
c
Positive control.
have been fully assigned with the aid of 1H-1H COSY and 1H-13
C
HSQC spectra. To our surprise, the other hexavalent conjugate 24
showed significantly differences in NMR spectra from its analogs
(18, 27 and 30). The 1D NOE and 2D-ROESY spectra of 24 clearly
indicated that there was conformational exchange in solution (SI
Figs. 1 and 2).
the weakest activity with IC50 over 100 mM. In the case of b-CD
derivatives (19, 25, 28 and 31), the effect of the four pentacyclic
triterpenes on the anti-influenza A/WSN/33 virus activity was in
the order: EA > UA > OA > BA. But, interestingly, the relationship
was reversed for
exhibited the most potent anti-influenza A/WSN/33 virus activity
with an IC50 of 4.7 0.52 M, a CC50 > 100 M and a selectivity
a-CD and g-CD derivatives. Compound 28
2.2. Biological assays
m
m
index (SI) of >21.3 (Table 1 and Fig. 3). Another compound 31 also
showed strong anti-influenza virus activity with an IC50 of
6.5 1.24 mM and a selectivity index (SI) of >15.4.
Compared with our previous work on pentacyclic triterpene-CD
conjugates [24], we can see clearly that the methylation or acety-
lation of C-2 and C-3 hydroxys of CD has significant effect on their
anti-influenza virus activity. The acetylation decreased the antiviral
activity by ~1.4e4.2 fold, while the methylation decreased the
2.2.1. Anti-influenza virus activity assay
Antiviral activity of the newly synthesized multivalent per-(2,3-
di-O-methyl)-a-, b- or g-CD-based pentacyclic triterpene conju-
gates (18e20 and 24e32) was tested against influenza A/WSN/33
virus that was propagated in MDCK cells by the CellTiter-Glo assay
and the cytopathic effect (CPE) reduction assay [33]. As shown in
Table 1, no significant cytotoxicity was observed at the concentra-
tion of 100
relationship between anti-influenza A/WSN/33 virus activity and
CD scaffold was similar to our previously results [24], i.e., -CD is
mM for all the conjugates. Except compound 29, the
potent by ~1.7e8.9 fold, even with two
g-CD conjugates diminished
activity (IC50 > 100 M), indicating that the alkyl of the hydroxyls of
m
b
CD conjugates will decrease their antiviral activity.
The inhibitory activity of compound 28 against influenza A/
WSN/33 virus was confirmed by direct observation under a
the best candidate for the construction of the multivalent conju-
gates and the smaller or larger CDs would decrease the anti-
influenza virus activity. Two g-CD derivatives (26 and 32) showed
Fig. 2. Characteristic portions of the 400-MHz 13C NMR spectra of 18, 27 and 30 recorded in CDCl3 at 298K.

Z. Tian et al. / European Journal of Medicinal Chemistry 134 (2017) 133e139
137
28. The results showed that the production of progeny virus was
markedly decreased when compound 28 was present during the
period from 0 to 2 h (entry stage) or from 0 to 10 h (the whole virus
lifecycle), as compared with DMSO control, indicating that com-
pound 28 exhibited the inhibitory effect at the early stage of virus
infection (0e2 h) (SI Fig. 4). On the contrary, compound 28 had no
inhibitory effects on viral production when added 2 h post-
infection or later. Based on these data, we concluded that com-
pound 28 was only effective at viral entry step [24].
The transmembrane viral envelope protein HA plays a crucial
role at the early stage of influenza virus infection. Therefore, we
investigated whether compound 28 targeted HA protein using the
hemagglutination inhibition (HI) assay. In this assay, three-fold
serial dilutions of compound 28 were made from 0.4 to 10 mM. In
the RBC-only control, the RBCs did not agglutinate (Fig. 4A, lane 1,
lower row). When RBCs were mixed with influenza virus, hemag-
glutination was detected (Fig. 4A, lane 1, upper row). Inhibition of
hemagglutination was observed for compound 28 at concentra-
Fig. 3. Inhibition curve of compound 28. Concentrations of 28 were 0.625
2.5 M, 5 M, 10 M, and 20 M. Each concentration was tested in triplicate, and the
results are expressed as the mean and standard deviation.
mM, 1.25 mM,
tions of 0.4e10
upper row). In parallel, inhibition of hemagglutination was also
observed for anti-HA antibody at concentrations of 0.1e2.7 g/mL
mM in a dose-dependent manner (Fig. 4A, lane 2e5,
m
m
m
m
m
(Fig. 4B). The HI assays implied that compound 28 and anti-HA
antibody have the same target, HA. Surface plasmon resonance
(SPR) experiments were subsequently performed with a Biacore
200 optical biosensor to determine the binding affinity of com-
pound 28 with HA. As shown by SPR responses in Fig. 4C, com-
pound 28 showed specific binding to HA protein immobilized on
the CM5 chip and exhibited dose-dependent responses. The
calculated equilibrium dissociation constant (KD) values for
microscope, which showed that 28 significantly reduced the CPE
induced by influenza virus infection compared with the DMSO
control (SI Fig. 3).
2.2.2. Mechanistic exploration
To explore the mechanism of the antiviral effect of compound
28, we firstly used the time-of-addition assay to estimate which
stage(s) of the influenza virus life cycle was inhibited by compound
Fig. 4. Interactions of HA with compound 28. (AeB). HI assay was performed to compare compound 28 vs. anti-HA antibody in inhibiting influenza virus-induced aggregation of
chicken erythrocytes. (C). SPR assay to determine the affinity of compounds 25 and 28 to HA protein immobilized on a CM5 sensor chip. The KD values of compounds 25 and 28 are
9.7 and 4.0 mM, respectively.

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Z. Tian et al. / European Journal of Medicinal Chemistry 134 (2017) 133e139
Fig. 5. A proposed mechanism for multivalent pentacyclic triterpene-functionalized per-O-methylated CD conjugates mediated blocking of influenza virus entry, wherein the
multivalent pentacyclic-triterpenes tightly bind to viral homotrimeric HA protein, thus disrupting the attachment of viruses to host cells.
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compound 28 showed higher affinity to HA protein than its analog
25.
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In the present study, we designed and synthesized a series of
pentacyclic triterpene-functionalized per-(2,3-di-O-methylated)
- and -CD conjugates via click chemistry and tested their anti-
influenza A/WSN/33 (H1N1) virus activity in MDCK cells. No sig-
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for all the conjugates in MDCK cells. Two conjugates 28 and 31
exhibited the highest anti-influenza virus activity with IC50 at
micromole level and SI > 15. Mechanism investigation indicated
that they showed high affinity to influenza HA protein with KD at
mM level, thus disrupting the interaction of influenza HA with the
SA-linked glycoprotein receptor (Fig. 5). This work, as well as our
previous study on multivalent pentacyclic triterpene-CD conju-
gates [24], provides the basis for the design of multivalent com-
pounds based on CD scaffold as anti-influenza virus entry inhibitors
and might pave the way for a variety of practical applications for
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grants Nos. 81573269, 81373271, 21572015
and 81361168002). We thank Dr. Zhong at the Centre of Medical
and Health Analysis, Peking University for MALDI-TOF experiments.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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