Structure-Based Tetravalent Zanamivir with Potent Inhibitory Activity against Drug-Resistant Influenza Viruses

Article abstract of DOI:10.1021/acs.jmedchem.6b00537

Zanamivir and oseltamivir are principal influenza antiviral drugs that target viral neuraminidase (NA), but resistant viruses containing mutant NAs with diminished drug affinity are increasingly emerging. Using the structural knowledge of both drug-binding sites and their spatial arrangement on the homotetrameric NA, we have developed a tetravalent zanamivir (TZ) molecule that exhibited marked increases in NA binding affinity, inhibition of NA enzyme activity, and in vitro plus in vivo antiviral efficacy over zanamivir. TZ functioned against both human seasonal H3N2 and avian H7N9 viruses, including drug-resistant mutants. Crystal structure of a resistant N9 NA in complex with TZ explained the function, which showed that four zanamivir residues simultaneously bound to all four monomers of NA. The design method of TZ described in this study may be useful to develop drugs or ligands that target proteins with multiple binding sites. The potent anti-influenza activity of TZ makes it attractive for further development.

Full text of DOI:10.1021/acs.jmedchem.6b00537

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Article
Structure-based Tetravalent Zanamivir with Potent
Inhibitory Activity against Drug-resistant Influenza Viruses
Lifeng Fu, Yuhai Bi, Yan Wu, Shanshan Zhang, Jianxun Qi, Yan Li, Xuancheng
Lu, Zhenning Zhang, Xun Lv, Jinghua Yan, George F. Gao, and Xuebing Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00537 • Publication Date (Web): 24 Jun 2016
Downloaded from http://pubs.acs.org on June 25, 2016
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Structure-based Tetravalent Zanamivir with Potent Inhibitory
Activity against Drug-resistant Influenza Viruses
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Lifeng Fu,†,‡,∇,⊥ Yuhai Bi,†,⊥ Yan Wu,†,⊥ Shanshan Zhang,†,∇ Jianxun Qi, Yan Li, Xuancheng Lu,
†
†
§
†
,∇
†
†,#
,†, #
,†,‡,#
Zhenning Zhang, Xun Lv, Jinghua Yan, George F. Gao,* and Xuebing Li*
†
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,
Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China
‡
National Engineering Research Center for Carbohydrate Synthesis, Jiangxi Normal University,
Nanchang 330022, China
∇Graduate University of Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China
§
Laboratory Animal Center, Chinese Center for Disease Control and Prevention, Changping District,
Beijing 102206, China
#
Center for Influenza Research and Earlyꢀwarning, Chinese Academy of Sciences (CASCIRE),
Chaoyang District, Beijing 100101, China
ABSTRACT: Zanamivir and oseltamivir are principal influenza antiviral drugs that target viral
neuraminidase (NA), but resistant viruses containing mutant NAs with diminished drug affinity are
increasingly emerging. Using the structural knowledge of both drugꢀbinding sites and their spatial
arrangement on the homotetrameric NA, we have developed a tetravalent zanamivir (TZ) molecule
that exhibited marked increases in NA binding affinity, inhibition of NA enzyme activity, and in
vitro plus in vivo antiviral efficacy over zanamivir. TZ functioned against both human seasonal
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H3N2 and avian H7N9 viruses, including drugꢀresistant mutants. Crystal structure of a resistant N9
NA in complex with TZ explained the function, which showed that four zanamivir residues
simultaneously bound to all four monomers of NA. The design method of TZ described in this study
may be useful to develop drugs or ligands that target proteins with multiple binding sites. The potent
antiꢀinfluenza activity of TZ makes it attractive for further development.
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INTRODUCTION
Influenza neuraminidase (NA) is a homotetrameric enzyme on the viral envelope, and hydrolyzes
sialic acids from glycan receptors on the host cells to enable the release and spread of progeny virus
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from the infected cells. Antiꢀinfluenza drugs, zanamivir (ZNV) and oseltamivir (OSV), are two
structural analogues of sialic acid in the catalytic transition state (Figure 1) that interact with the NA
2,3
active site more potently than the natural substrate, thereby reducing virus propagation. As the
amino acid residues within and near the active site are highly conserved, ZNV and OSV are effective
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against all influenza A and B viruses. However, drugꢀresistant mutations have been increasingly
identified in the NA of various strains, including human seasonal H3N2 and H1N1 viruses, and avian
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H5N1 and H7N9 viruses that are capable of infecting humans.
Evidence suggests that the
mutations compromise the interaction between NA and drugs, although they retain the ability to
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engage the active site.
Functional group modification of ZNV or OSV
and exploration of a
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structurally new NA inhibitor
may antagonize drug resistance. Synthetic conjugates that contain
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multiple copies of an inhibitor displayed on appropriate scaffolds,
as typically illustrated by a
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series of divalent zanamivir derivatives,
can also increase antiviral activity. However, because
the multivalent inhibitors described so far were designed without consideration of spatial
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arrangement of the active sites on the tetrameric NA molecules, the activity gain is largely a
consequence of their ability to induce virus aggregation on the host cell surface, rather than the cause
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of an increase in the intrinsic binding affinity.
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RESULTS AND DISCUSSION
The crystal structures of NA show that four active sites are symmetrically arranged on the top
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surface of the NA tetramer with a spacing of ∼38 Å from the center of symmetry.
The exposed
radial arrangement of the sites is thermodynamically favorable for oligovalentꢀligand bindings, as
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seen for the pentameric Shigaꢀlike toxin
and the trimeric adenovirus fiber protein. On the basis
of these findings, we designed a set of conjugated molecules, in which four terminal ZNV residues
were anchored on a small central scaffold by oligoethylene glycol (OEG) linkers of different lengths
(Figure 1D). We anticipated that compound 1, with a molecular dimension complementary to the
distribution of NA binding sites, might occupy all the sites of a NA molecule and therefore
dramatically improve its intrinsic binding affinity through the decreased binding energy. To develop
a putatively potent NA inhibitor, we took into account three essential parameters when designing the
TZs. First, an easily accessible and chemically symmetric tetraazide (16, Scheme 1) served as the
scaffold, from which four ZNV residues radiated out to engage all NA subunits. Second, ZNV but
not OSV was chosen as the pharmacophore, because ZNV is structurally more similar to the natural
1
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NA substrate, and therefore the viral resistance to ZNV occurs less frequently. In addition, all
functional groups around the cyclohexene core of OSV are involved in the interaction with the NA
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active site, whereas in the enzymeꢀbound state, the 7ꢀhydroxyl group of ZNV is oriented toward the
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outside of the site and makes no virtual contact with the enzyme. This provided the best connection
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point for tethering ZNV pharmacophores with minimized impairment of the binding affinity. Finally,
a flexible OEG linker (9, 10, or 11) was used to bridge the pharmacophores and the scaffold, with its
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beneficial pharmacological properties contributing to the resistance to nonspecific binding and
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metabolic clearance, as well as the aqueous solubility of the large TZs.
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Using the known crystal structure of an N9 NA, our molecular modelling study predicted that
compound 1 would attach to the protein surface, through the four ZNV residues binding
simultaneously to the active sites (Figure 1E). The calculated free energy of 1 binding to NA (−111
−1
−1
kcal mol ) is markedly lower than that of ZNV binding (−58 kcal mol ), suggesting that a
substantial affinity increase may be achieved using such a complemented oligovalent interaction. We
then synthesized 1, as well as the controls 2 and 3, to perform functional and mechanistic studies. A
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concise modular strategy that accommodated the aboveꢀmentioned design elements was used to
yield synthetic assemblies. Nucleophilic substitution of a 7ꢀactivated ZNV derivative (12) by an
amineꢀterminated OEG (9, 10, or 11) accomplished coupling between the pharmacophore and the
linker. The alkyne functionality at the other end of the linker allowed the straightforward installation
of the pharmacophores onto the scaffold 16 through the use of a Cu(I)ꢀcatalyzed Huisgen
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cycloaddition reaction. All resulting TZs showed excellent aqueous solubility (≥1.0 M), and were
fully characterized by HPLC (purity > 95%), NMR, and mass spectroscopy.
We cloned four recombinant influenza NAs: a N2 and its mutant (mutN2) from H3N2
A/Moscow/10/99, and two N9s from H7N9 A/Anhui/1/2013 (anhN9) and H7N9 A/Shanghai/2/2013
(
shaN9), respectively. These soluble proteins were prepared through a baculovirus−insect cell
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expression system
and were used as models to examine the interactions with TZs. The N2 is
a common NA serotype found in seasonal and pandemic H3N2 viruses and the N9 represents a
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serotype found in emerging avian H7N9 viruses that can cause fatal infection in humans. The mutN2
carries E119V and I222L dual substitutions (N2 numbering) and the shaN9 has an R294K
replacement relative to anhN9 (N9 numbering). These mutations lie within the NA active site and are
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known to confer resistance to OSV and ZNV.
measure the inhibition of NA enzyme activity, all TZs exhibited potent efficacies for the test NAs
Figure 2A−D). In each case compound 1 showed a higher activity than other TZs and this
In the assays using a fluorescent substrate
to
(
superiority became more evident for the inhibition of drugꢀresistant mutN2 and shaN9. Similarly,
compound 1 had a similar efficacy to ZNV for the inhibition of the drugꢀsensitive N2 and anhN9,
whereas for the resistant NAs, it retained a lowꢀ or subꢀnanomolar potency that was at least 6ꢀfold
higher than that of ZNV. Additionally, an octavalent ZNV conjugate (25, Scheme 2), which has a
central scaffold and OEG linkers similar to those of TZs, was less effective than compound 1 for the
inhibition of all tested enzymes. This result demonstrates that the increased multivalency could not
result in further improvement in the activity.
Surface plasmon resonance (SPR) experiments showed that TZs and ZNV bound to both N2
and mutN2 NAs, with the responses proportional to their molecular masses (Figure 2E). In contrast,
no interaction was detected between TZs and bovine serum albumin (BSA) or between the NAs and
a tetravalent OEG backbone molecule (29) that contained no ZNV residues (for the structure of 29,
see Scheme 2; for the SPR sensorgrams, see Figure S1 in the Support Information). This confirmed
that the ZNV residues of TZs were responsible for the observed specific binding. In almost all cases,
TZs showed slightly slower association rates (kon) than ZNV, consistent with the increased molecular
size that hampers motility. TZs also showed reduced dissociation rates (koff). In particular, the
decreases in the koff value of 1 were significant, which resulted in an approximate picomolar affinity
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(
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) for both NAs, strongly suggesting that this molecule could interact with NAs to form highly
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stable complexes. As the known divalent
and tetravalent
ZNV derivatives, of which the
molecular dimension does not match the distribution of NA active sites, generally showed a lower
NA affinity (or NAꢀinhibitory activity) than ZNV, the binding kinetic analysis of compound 1
further reinforce the hypothesis that a marked increase in affinity could be generated through a
complemented multiple interaction. In addition, although it is difficult to directly correlate the
binding kinetics to the enzyme inhibition of TZs because of the different assay formats, the
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enhancement in both the affinity (K ) and efficacy (IC50) of 1 compared to those of ZNV were more
markedly observed for the drugꢀresistant NA than the sensitive NA.
We solved the crystal structure of shaN9 NA in complex with compound 1 to explore the
structural basis for the improved efficacy and affinity. The crystal diffracted to high resolution with
clear electron density showing that four ZNV residues connected to a short part of the linker were
trapped on the tetramer surface (Figure 2F and Figure S2). All the ZNV residues interacted with the
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NA active sites in an identical manner to that of ZNV bound by the same enzyme (Figure S3), and
the multiple bindings did not disrupt the tetramer structure. However, as the central part of the linker
and the scaffold were undetectable by crystallography owing to their flexibility and incompetence in
making direct contact with the enzyme, it was unclear whether these ZNV residues belong to a single
tetravalent molecule. To address this issue, we performed analytical ultracentrifugation (AUC)
experiments and revealed that the addition of compound 1 at different concentrations to the shaN9
tetramer in solution did not result in apparent intermolecular crossꢀlinks (Figure 3). This indicated
that the observed complex by crystallography was formed through a 1:1 binding of 1 to the shaN9
tetramer, and that the formation of this complex should be thermodynamically more preferable than
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that of intermolecular bridges. These findings explain the mechanism behind the remarkable ability
of compound 1 to bind and inhibit the enzyme.
To evaluate the therapeutic potential of compound 1, we examined its antiviral activity in
MadinꢀDarby canine kidney (MDCK) cells. Four viruses, which contain N2, mutN2, anhN9, and
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shaN9 NAs, respectively, were rescued with reverse genetics
and were used for assays
(
Table 1). In agreement with its IC50 against enzymes (Figure 2A−D), compound 1 inhibited viral
replication more potently than ZNV, with efficacies (EC50) in the lowꢀnanomolar range even against
the drugꢀresistant viruses. To ensure that the improved antiviral activity did not simply result from
the increased multivalency or steric hindrance, compounds 25 and 29 were tested as controls.
Octavalent 25 had a similar efficacy to 1, and the backbone molecule 29 showed no antiviral effect.
In addition, transmission electron microscopy (TEM) examination of the H3N2 virus after incubation
with 1 demonstrated that the tetravalent inhibitor was unable to induce viral aggregation (Figure 4
and Figure S4). This is in marked contrast to the divalent ZNV derivative that can bridge virus
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particles through interviral NA bindings.
The different binding modes of these two inhibitors
could be attributed to their structural features. The divalent compound, which has an 18ꢀatom linker
corresponding to ∼23 Å in length) to join two ZNV residues, is incapable of simultaneous
(
engagement to multiple sites within a NA molecule and, therefore, the binding to the NAs on
different virions occurs. This mechanism of action is also proposed to contribute to the increased
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antiviral activity of the known tetravalent ZNV derivatives.
cell cultures to the inhibitors is influenced by multiple factors besides NA, such as viral fitness and
assay process. This makes it difficult to clearly explain how the measured values of IC50 and K
Figure 2) can be translated to those of EC50 (Table 1). However, the TEM result, together with the
The sensitivity of virus replication in
D
(
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crystallographic and AUC studies, suggested that the potent in vitro antiviral activity of 1 is
associated with its strong binding to a single NA molecule on the viral surface.
We next assessed the efficacy of compound 1 in mice that were challenged with a
laboratoryꢀadapted influenza viral strain, A/Puerto Rico/8/34 (H1N1). A preliminary test to evaluate
the in vitro effect of inhibitors against this virus in a MDCK cell culture, showed that 1 exhibited an
activity lower than 4 nM, which was more than 25 times as potent as ZNV (Figure S5). In addition,
compound 1 demonstrated a mean halfꢀlife (T1/2) of 72 min in an intravenous pharmacokinetic study
in rats (Table S4). Although the rapid clearance of 1 from the circulation was over 2ꢀfold longer than
ZNV (T1/2 = 32 min), this convinced us that an oral or subcutaneous route of administration was
unlikely to provide efficient protection to mice against a lethal viral infection. The infected mice
were therefore treated intranasally with the inhibitors, in a similar manner to the inhaled ZNV in
clinical use, which delivers the drug directly to the site of infection (respiratory tract). The first set of
experiments used a multiple dosage regimen, in which the inhibitors were administered once daily
for 7 consecutive days (Figure 5A and C). Compound 1 had a superior effect to ZNV at doses of 5
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and 10 mg kg and protected all mice from lethal virus challenge. In another experimental set that
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used a singleꢀdose administration, all animals receiving ZNV at 20 mg kg died within 9 days
(Figure 5B and D). In contrast, the single treatment of 1 at an equivalent dosage provided 100%
protection, demonstrating its prolonged effect. Pathological examination of mouse lung tissues at 7
days after infection indicated the pathology results correlated well with the antiviral treatments.
Severe histological changes associated with bronchointerstitial pneumonia were detected in the
untreated (Figure 5I) and uncured (Figure 5K and L) animals, whereas no appreciable lesions were
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observed in compound 1ꢀtreated mice that survived (Figure 5F and H) or in healthy controls (Figure
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CONCLUSIONS
The superior in vivo efficacy of compound 1 over ZNV could be attributed to its improved
binding with viral NA, and probably also to the increased molecular size that prolongs its residency
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in the respiratory tract, as demonstrated by the divalent
and tetravalent ZNV derivatives. The
substantial increase in binding affinity, which arises from the simultaneous engagement of the
inhibitor to all four active sites of the enzyme as designed, is important because it results in the
efficient inhibition of both drugꢀsensitive and drugꢀresistant NAs. mutN2 and shaN9 used here
contain either the E119V/I222L or R294K substitution that compromises the NA affinity of the
12,43
drugs.
For example, the E119V mutation disrupts binding with the 4ꢀguanidine of ZNV or the
4ꢀamine of OSV, and R294K reduces the interaction with the carboxylate of both ZNV and OSV.
The high activity of 1 against these mutant NAs demonstrates that the enhanced binding affinity
compensated for the loss of interactions. Currently, drugꢀresistant strains with similar mutations to
5−8
those used in this study are emerging. Given the pandemic threat of resistant viruses, in addition to
the fact that ZNV and OSV are the only two drugs recommended by the US Food and Drug
Administration for worldwide use, the potent NA inhibitor 1, with extended antiviral activities in
both cell culture and animal models, represents a potential new treatment for influenza infection that
may also be effective in controlling the current drugꢀresistant viral mutants. The success in targeting
this tetravalent molecule to the surface of an enzyme tetramer suggests that this inhibitor design
might be generally applicable to many proteins with multiple actives sites.
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EXPERIMENTAL SECTION
General Chemistry Information. All chemical regents and solvents, including ZNV and OSV
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(in the form of oseltamivir carboxylate), were obtained from commercial source and used as
purchased. TZs (1−3), 25, and 29 were synthesized as described below. Other intermediate
compounds were synthesized as described in the Support Information (in the section of Synthetic
Protocols). Silica gel (100−200 mesh) for column chromatography was purchased from Qingdao
1
Haiyang Chemical Co., Ltd (China). Sephadex Gꢀ15 was purchased from GE Healthcare (China). H
13
NMR (500 MHz) and C NMR (125 MHz) spectra were measured on a Bruker Advance
spectrometer. ESIꢀMS and ESIꢀHRMS spectra were recorded on a Bruker Apex IV FTMS
spectrometer. The purity of final compounds is ≥95%, which was determined by an HPLC system
(Agilent Technologies 1200) equipped with a UV detector using conditions as follows: Atlantis
HILIC Silica columns (250 × 4.6 mm, 5 ꢁm), 95→40%A (solvent A: 25% aqueous ammonia
solution/acetonitrile (99:1, v/v), solvent B: 25% aqueous ammonia solution/deionized water (99:1,
v/v)) gradient at 1 mL min−1 over 25 min, UV detection at 220 nm wavelength. The HPLC
chromatograms for the analysis of TZs and compound 25 are provided in the Supporting Information
(in the section of HPLC Chromatograms).
Tetravalent Zanamivir Conjugate (1). To a solution of compound 17 (120 mg, 0.024 mmol)
in MeOH (2 mL) was added an aqueous solution of NaOH (1 M, 1 mL). The mixture was stirred at rt
+
for 3 h, neutralized with Dowex 50 (H ), filtered, and concentrated. The residue was dissolved in
2 2
CH Cl /TFA (2 mL, 1:1, v/v). The mixture was stirred at rt for 3 h and concentrated. The resulting
residue was dissolved in deionized water (0.5 mL) and filtered (Millipore Express 0.22 ꢁM filter).
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Purification with Sephadex Gꢀ15 (eluent: 0.1% TFA in H
2
O) yielded 1 as a white solid (65.3 mg,
2
0
1
6
8
8%). mp 190−210 °C (with decomposition); [α]
D
+28.6 (c 0.8, H
2
O); H NMR (500 MHz, D
2
O): δ
.16 (s, 4H, 4−HC=C−), 5.86 (d, J = 2.2 Hz, 4H, 4Hꢀ3), 4.86 (d, J = 8.9 Hz, 4H), 4.60 (s, 8H), 4.56
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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43
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45
46
47
48
49
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51
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55
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57
58
59
60
(
s, 8H), 4.47 (d, J = 9.9 Hz, 4H), 4.35 (dd, J = 8.8, 2.0 Hz, 4H), 4.05 (t, J = 9.4 Hz, 4H), 3.93−3.90
CH
m, 4H), 3.63−3.56 (m, −OCH O−, 192H), 3.40−3.38 (m, 4H), 3.19−3.10 (m, 8H), 1.87 (s, 12H,
NAc); C NMR (125 MHz, D O): δ 173.8, 162.6, 156.9, 144.9, 128.0, 117.1, 115.1, 109.2, 75.5,
(
2
2
13
4
7
2
2
294 32
1.6, 69.6, 69.3, 68.7, 60.4, 47.1, 40.4, 22.1; ESIꢀHRMS: m/z calculated for C165H N O80 [M +
2+
H] : 2001.9955, found: 2001.9908.
Tetravalent Zanamivir Conjugate (2). Using a procedure similar to that employed for the
synthesis of 1, deprotection of compound 18 (180 mg, 0.045 mmol) gave 2 as a white solid (103.4
20
1
mg, 78%). mp 180−200 °C (with decomposition); [α]
O): δ 8.17 (s, 4H, 4−HC=C−), 5.94 (d, J = 2.5 Hz, 4H, 4Hꢀ3), 4.88 (dd, J = 8.9, 2.2 Hz, 4H), 4.61
(s, 8H), 4.57 (s, 8H), 4.50 (dd, J = 9.9, 2.1 Hz, 4H), 4.36 (dd, J = 8.9, 2.4 Hz, 4H), 4.06 (t, J = 9.4 Hz,
H), 3.95−3.90 (m, 4H), 3.65−3.48 (m, 96H, −OCH CH O−), 3.43−3.39 (m, 4H), 3.26−3.10 (m, 8H),
D 2
+27.4 (c 0.8, H O); H NMR (500 MHz,
D
2
4
2
2
13
1.88 (s, 12H, 4NAc); C NMR (125 MHz, D
2
O): δ 170.8, 162.0, 156.6, 152.8, 145.0, 144.6, 127.0,
1
09.8, 108.9, 83.6, 77.1, 70.6, 66.1, 64.4, 52.6, 41.1, 28.0; ESIꢀHRMS: m/z calculated for
+
C
117
H
198
N
32
O
56 [M + 2H] : 1473.6809, found: 1473.6816.
Tetravalent Zanamivir Conjugate (3). Using a procedure similar to that employed for the
synthesis of 1, deprotection of compound 19 (70 mg, 0.02 mmol) gave 3 as a white solid (36.7 mg,
20
1
7
6%). mp 160−180 °C (with decomposition); [α]
D 2 2
+26.3 (c 0.8, H O); H NMR (500 MHz, D O): δ
8.16 (s, 4H, 4−HC=C−), 5.86 (s, 4H, 4Hꢀ3), 4.86 (d, J = 8.5 Hz, 4H,), 4.60 (s, 8H), 4.56 (s, 8H), 4.47
(
d, J = 10.2 Hz, 4H), 4.35 (d, J = 8.3 Hz, 4H), 4.05 (t, J = 9.4 Hz, 4H), 3.93−3.90 (m, 4H), 3.63−3.56
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(
m, 40H, −OCH
2
CH
2
O−), 3.48 (s, 8H), 3.40−3.38 (m, 4H), 3.19−3.10 (m, 8H), 1.87 (s, 12H, 4NAc);
1
3
2
C NMR (125 MHz, D O): δ 173.8, 165.2, 163.4, 157.0, 156.4, 145.5, 143.6, 127.8, 117.2, 114.9,
1
08.6, 75.7, 69.5, 69.3, 69.0, 68.7, 63.0, 62.4, 51.3, 50.1, 47.1, 40.1, 21.8; ESIꢀHRMS: m/z
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8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
calculated for C H N O [M + 2H] : 1209.5236, found: 1209.5210.
9
3
150 32 44
3
6
Octavalent Zanamivir Conjugate (25). Click coupling between compounds 24 and 14,
followed by a deprotection reaction, yielded 25. Specifically, to a solution of compound 24 (10 mg,
8
.6 µmol) and compound 14 (72 mg, 78 µmol) in THF/H
aqueous solution of CuSO (1 M) under a nitrogen atmosphere, followed by quick addition of sodium
ascorbate (3.96 mg, 0.02 mmol). The mixture was stirred at 40 °C for 3 h and concentrated. The
resulting residue was purified by silica gel column chromatography (CH Cl /MeOH 7:1, v/v) to give
an intermediate compound as a white solid (30 mg, 3.5 µmol, 45%). Deprotection of this compound
by a procedure similar to that employed for the synthesis of 1 gave 25 as a colorless oil (20.7 mg, 90%
2
O (1.5 mL, 2:1, v/v) was added a drop of
4
2
2
2
0
1
from intermediate compound, 37% from 24); [α]
.91 (s, 8H, 8−HC=C−), 5.51 (s, 8H, 8Hꢀ3), 4.74 (dd, J = 9.3, 1.5 Hz, 8H), 4.55 (s, 8H), 4.49 (s,
16H), 4.31 (m, 16H), 4.22 (dd, J = 9.2, 1.9 Hz, 8H), 3.88 (t, J = 9.8 Hz, 8H), 3.85 (m, 8H), 3.60−3.35
m, 228H, −OCH CH O−), 3.30 (dd J = 11.7, 6.6 Hz, 20H), 3.23 (d, J = 17.5 Hz, 16H), 3.12 (m, 8H),
3.06 (m, 8H), 1.77 (s, 24H, 8NAc); C NMR (125 MHz, D
43.9, 143.1, 126.1, 124.1, 105.0, 76.7, 75.2, 69.5, 69.3, 69.1, 68.9, 68.5, 51.6; ESIꢀHRMS: m/z
D 2 2
+36.8 (c 0.8, H O); H NMR (500 MHz, D O): δ
7
(
2
2
13
2
O): δ 173.8, 167.8, 156.9, 156.7, 148.6,
1
+
calculated for C H N O Na [M + Na] : 6580.0905, found: 6582.0822.
2
65 453 64 128
Tetravalent OEG Conjugate (29). Using a procedure similar to that employed above in the
click coupling reaction, compound 29 was afforded from 28 (131.8 mg, 0.28 mmol) and 16 (13 mg,
1
0
.056 mmol) as a white solid (90.6 mg, 77%). H NMR (500 MHz, CDCl
3
): δ 8.14 (s, 4H,
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2 2
−HC=C−), 4.60 (s, 8H), 4.28 (s, 8H), 3.55−3.46 (m, 137H, −OCH CH O−), 3.44 (m, 8H), 3.28 (s,
13
2H, 4CH
3
O); C NMR (125 MHz, CDCl
3
): δ 144.8, 144.7, 131.0, 128.5, 126.8, 77.5, 77.2, 77.0,
+
1.8, 70.5, 69.9, 58.9; ESIꢀHRMS: m/z calculated for C93
H
177
N
12
O
40 [M + H] : 2102.2180, found:
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102.2178.
NAs, Viruses, Cells, and Animals. All NAs (N2, mutN2, shaN9, anhN9) were cloned,
1
2,19,37−42,48
expressed, and purified as previously described.
For mutN2 that contained E119V and
I222L, point mutations were introduced into the gene encoding the wildꢀtype N2 (Generay Biotech,
12,42,44−46
China). All viruses were generated using reverse genetics as previously described.
The
A/Puerto Rico/8/34 (H1N1) virus was rescued from the genes of the natural isolate. The H3N2 virus
contained the hemagglutinin gene and the NA gene of A/Moscow/10/99 (H3N2) with the remaining
genes from A/Puerto Rico/8/34 (H1N1). The mutH3N2 virus had similar genes to the H3N2 virus,
except for the mutant NA gene carrying E119V and I222L. The anhH7N9 and shaH7N9 viruses were
produced from the genes of A/Anhui/1/2013 (H7N9) and H7N9 A/Shanghai/2/2013 (H7N9),
respectively. MDCK cells were propagated in DMEM (Dulbecco’s modified Eagle medium)
−1
−1
containing 10% fetal calf serum, 100 U mL penicillin, and 100 U mL streptomycin, and
49
maintained following known protocols. The rescued viruses were amplified in MDCK cells. Viral
culture supernatants were harvested, concentrated, and stored at −80 °C. The pfu and the TCID50 (50%
tissue culture infective dose) of viruses were quantified in MDCK cells. All experiments with the
viruses were performed in approved biosafety level (BSLꢀ3) containment laboratories. BALB/c mice
and SpragueꢀDawley rats were used for infection protection and pharmacokinetic experiments,
respectively. Both were female and seven weeks old, and purchased from Vital River (China). All
animals were used according to approved protocols for their care and use.
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Computer Modeling. The known crystal structure of N9 NA in complex with ZNV (PDB
12
code: 4MWX) was used for computer simulation. The 3D structure of compound 1 was initially
generated with ChemBio3D Ultra (version 11.0) and refined by running energy minimization with
MOE (molecular operating environment, version 2014.09). The docking of 1 to N9 tetramer was
carried out using MOE with standard program settings. Following the energy minimization of 1−N9
complex, the free binding energy was calculated using a GBVI/WSA (generalizedꢀborn volume
integral/weighted surface area) program in MOE. The free energy of ZNV binding to N9 monomer
was also calculated similarly. Figure 1E was created with PyMOL (version 1.7.4).
0
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3
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8
9
0
NA Inhibition Assay. NA enzyme activity and competitive inhibition by inhibitor (OSV, ZNV,
TZs, or compound 25) were tested using MUNANA (4ꢀmethylumbelliferylꢀNꢀacetylneuraminic acid)
12,19,38,50
as a fluorescent substrate, according to published protocols.
In brief, NA activity was
predetermined in an appropriate range (normally in 10−50 nM, in PBS) for fluorescence detection in
the presence of MUNANA (167 ꢁM, in PBS). For inhibition assays, the enzyme (10 ꢁL) was mixed
with serial 5ꢀfold dilutions of inhibitor (10 ꢁL, initial concentration at 1 mM) in PBS in a 96ꢀwell
microplate and incubated at 37°C for 30 min. MUNANA (30 ꢁL, 167 ꢁM, in PBS) was then added to
the mixture and fluorescence was measured on a SpectrMax M5 microplate reader (Molecular
Devices), at excitation and emission wavelengths of 355 and 460 nm, respectively. Each assay was
performed in no less than triplicates. IC50 values were determined with GraphPad Prism (version
5.0).
SPR Assay. The binding of inhibitor (ZNV or TZs) to NA (N2 or mutN2) were analyzed by a
BIAcore T100 system (GE Healthcare, Sweden) based on SPR technique. NA was covalently
immobilized on a CM5 sensor chip (research grade) by an amineꢀcoupling method according to the
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manufacturer’s instructions. Five concentrations of inhibitor in PBS (10, 30, 90, 270 and 810 nM)
were preꢀprepared. The singleꢀcycle kinetics protocol was used for SPR analysis. To each cycle, a
solution of inhibitor was consecutively poured over the chip surface from low to high concentration
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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50
51
52
53
54
55
56
57
58
59
60
−
1
at a flow rate of 40 ꢁL min . BSA and compound 29 were tested as negative controls instead of NA
and inhibitor, respectively. Binding kinetic parameters were calculated using a steady affinity model
with the BIAcore T100 analysis software (version 2.0.1).
X-ray Crystallography. Crystals of shaN9 were obtained by the vapour diffusion method
12
from sitting drops as described previously. Compound 1−shaN9 complexes were prepared by
soaking shaN9 crystals in crystallization solution (0.1 M HEPES, pH 7.5, containing 5% v/v
2
ꢀmethylꢀ2,4ꢀpentanediol and 10% v/v PEG 10,000) supplemented with 50 mM 1 at 4 °C for 3 h.
The crystals were then flashꢀcooled at 100 K with cryoprotection. Diffraction data were collected on
the beamline BL17U at Shanghai synchrotron radiation facility. The data were processed and scaled
5
1
with HKL2000. The structure of shaN9 in complex with 1 was determined by molecular
52
replacement in Phaser using the CCP4 program suite, with the known N9 NA structure (PDB code:
5
3
54
7NN9) as a search model. Extensive model building was performed with Coot and refined with
55
56
Refmac. Further refinement was done with phenix.refine implemented in the Phenix package.
57
The stereochemical quality of final model was validated with Procheck. Figure 2F was produced
with PyMOL. Crystallographic data and refinement statistics are provided in Table S3.
AUC Analysis. A solution of shaN9 in PBS (4.2 µM, 400 ꢁL) was incubated at 37 °C for 3 h,
in the absence or presence of inhibitor (1 mM solution of ZNV or compound 1 in PBS, 1.6 ꢁL for 1
equiv assay and 9 ꢁL for 5 equiv assay). The mixtures were then transferred into 400 ꢁL
sectorꢀshaped 60Ti cells and scanned with a ProteomeLab XLꢀ1 protein characterization system
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(
Beckman Coulter), at 20 °C, 230 nm, and the rotor speed of 40,000 rpm. Absorbance data were
collected and analyzed with the SEDFIT software (version 9.4) to obtain the molecular weight
distribution of shaN9.
0
1
2
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9
0
In Vitro Viral Inhibition Assay. The in vitro antiviral activity of inhibitor (OSV, ZNV, 1, 25,
or 29) was determined in MDCK cell cultures. Serial 2ꢀfold dilutions of inhibitor (initial
concentration at 1 mM) in DMEM were prepared and mixed with an equal volume of virus (200
TCID50) in the same medium. Following incubation at 37 °C for 1 h, the mixtures (100 µL) were
4
added to MDCK cells, which were grown on 96ꢀwell microplates (10 cells per well). Further
incubations (37 °C, under a 5% CO atmosphere, 72 h) allowed for the inhibition of virusꢀinduced
2
cytopathic effects (CPEs) to be confirmed by microscopic examination. The virus titre in the infected
cells was then determined with hemagglutination test and the EC50 values were calculated with the
58,59
Reed and Muench method.
Four replicates were used for this viral inhibition assay.
Cytotoxicity Test. The cytotoxicity of inhibitor (OSV, ZNV, 1, 25, or 29) on MDCK cells was
60
measured using the standard procedure of MTT assay.
7
TEM Examination. The H3N2 virus in DMEM (150 µL, 10 pfu) was incubated at 37 °C for
3
h, in the absence or presence of inhibitor (10 µL, 1 mM solution of ZNV, 1, or 25 in PBS). The
4
6
mixtures were then processed for TEM analysis (JEOL 1400) as described previously.
Pharmacokinetic Analysis. SpragueꢀDawley rats (n = 5) were given a single intravenous
−
1
injection (in the tail vein) of inhibitor (ZNV or 1 in physiological saline) at a dosage of 6 ꢁmol kg .
Blood sample collection and processing, inhibitor concentration measurement, and pharmacokinetic
61
assessment were performed following the previously published method. The HPLC conditions used
to identify the inhibitors were as follows: (i) for ZNV: Ultimate C18/SCX column (5 ꢁm, 2.1 × 100
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mm), eluent A: 5 mM aqueous solution of ammonium acetate containing 0.1% v/v formic acid,
−1
eluent B: acetonitrile, 20→85%A gradient over 5 min at 0.6 mL min ; (ii) for compound 1:
ACQUITY CSH C18 column (1.7 ꢁm, 2.1 × 50 mm), eluent A: deionized water containing 1% v/v
ammonium hydroxide, eluent B: acetonitrile containing 1% v/v ammonium hydroxide, 10→95%A
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60
−
1
gradient over 5 min at 0.6 mL min .
Mice Protection Experiments. Under anaesthesia, six mice per group were intranasally
5
inoculated with the A/Puerto Rico/8/34 (H1N1) virus (10 pfu, in PBS, 40 µL). The inoculation was
performed by dripping viral suspension into the nasal passages of mice with pipettes. For
−
1
−1
multipleꢀdose antiviral treatment, mice were intranasally administered with 5 or 10 mg kg day of
the inhibitor (ZNV or 1 in PBS, 40 µL) for 7 days, starting at 1 day pi. For singleꢀdose antiviral
−
1
treatment, mice were treated only once with 20 mg kg of inhibitor (ZNV or 1 in PBS, 40 µL) at 2 h
−1
pi. The infected control group was treated with 40 µL day of PBS alone. All the treatments were
performed by dripping antivirals or vehicles into the nasal passages of mice with pipettes. The mice
(
n = 6) without viral inoculation were used as the healthy control group. Body weight and survival of
all mice were monitored for 14 days. Mice were accounted as dead when losing either 25% of their
initial weight or when they were moribund. Figure 5A−D were created with the GraphPad Prism
(version 5.0) on the basis of recorded data.
Pathological Examination. In mice protection experiments, one mouse per group was
euthanized at 7 day pi, and the lung was harvest for pathological examinations. After immersion in 4%
aqueous solutions of paraformaldehyde for 72 h, the excised tissues were processed for paraffin
embedding and cut into 5 ꢁmꢀthick sections, which were then stained using the standard
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haematoxylin and eosin procedure. Pathological changes were examined with a Leica DM2500
upright metallurgical microscope.
Statistics. In mice protection experiments, statistically significant differences between the data
of infected controls and those of inhibitorꢀtreated groups were determined using MantelꢀCox test in
GraphPad Prism (version 5.0).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Please insert doi number here.
Supplementary figures and tables, synthetic procedures and analytical data for intermediate
compounds, NMR and MS spectra, HPLC chromatograms (PDF)
Accession Codes
Atomic coordinates and structure factors for the crystal structures of shaN9 in complex with
compound 1 can be accessed using PDB codes: 5JYY. Authors will release the atomic coordinates
and experimental data upon article publication.
AUTHOR INFORMATION
Corresponding Authors
*(X. Li) Eꢀmail: lixb@im.ac.cn
*
(G. F. Gao) Eꢀmail: gaof@im.ac.cn
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Author Contributions
⊥These authors contributed equally.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors are grateful to Jinwei Ren (Institute of Microbiology, CAS) for technical help with the
NMR experiments; Yuanyuan Chen and Zhenwei Yang (Institute of Biophysics, CAS) for technical
help with the SPR experiments; Xiaoxia Yu (Institute of Biophysics, CAS) for technical help with
the AUC experiments; Jingnan Liang (Institute of Microbiology, CAS) for technical help with the
TEM analysis; Junfeng Hao and Guizhi Shi (Institute of Biophysics, CAS) for technical help with the
pathological examination in the animal study. This work was funded by the Ministry of Science and
Technology of China (973 program: 2012CB518803), the National Natural Science Foundation of
China (programs: 31470801, 81273381, 21202195, 81301465 and 81330082), the Development
Fund for Collaborative Innovation Center of Glycoscience of Shandong University, and the Open
Project Program of National Engineering Research Center for Carbohydrate Synthesis, Jiangxi
Normal University. Yan Wu is supported by Youth Innovation Promotion Association, CAS
(program number: 2016086).
ABBREVIATIONS USED
AUC, analytical ultracentrifugation; CPE, cytopathic effect; DMEM, Dulbecco’s modified Eagle
medium;
GBVI,
generalizedꢀborn
volume
integral;
HEPES,
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ꢀ(2ꢀhydroxyerhyl)piperazineꢀ1ꢀerhanesulfonic acid; MDCK, MadinꢀDarby canine kidney; MOE,
molecular operating environment; MTT, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium
bromide; MUNANA, 4ꢀmethylumbelliferylꢀNꢀacetylneuraminic acid; NA, neuraminidase; OEG,
oligoethylene glycol; OSV, oseltamivir; pi, postinfection; pfu, plaque forming units; SD, standard
deviation; SPR, surface plasmon resonance; TCID50, 50% tissue culture infective dose; TEM,
transmission electron microscopy; TZ, tetravalent zanamivir; WSA, weighted surface area; ZNV,
zanamivir
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