Discovery of Highly Potent Pinanamine-Based Inhibitors against Amantadine- and Oseltamivir-Resistant Influenza A Viruses

Article abstract of DOI:10.1021/acs.jmedchem.8b00042

Influenza pandemic is a constant major threat to public health caused by influenza A viruses (IAVs). IAVs are subcategorized by the surface proteins hemagglutinin (HA) and neuraminidase (NA), in which they are both essential targets for drug discovery. While it is of great concern that NA inhibitor oseltamivir resistant strains are frequently identified from human or avian influenza virus, structural and functional characterization of influenza HA has raised hopes for new antiviral therapies. In this study, we explored a structure-activity relationship (SAR) of pinanamine-based antivirals and discovered a potent inhibitor M090 against amantadine-resistant viruses, including the 2009 H1N1 pandemic strains, and oseltamivir-resistant viruses. Mechanism of action studies, particularly hemolysis inhibition, indicated that M090 targets influenza HA and it occupied a highly conserved pocket of the HA₂ domain and inhibited virus-mediated membrane fusion by locking the bending state of HA₂ during the conformational rearrangement process. This work provides new binding sites within the HA protein and indicates that this pocket may be a promising target for broad-spectrum anti-influenza A drug design and development.

Full text of DOI:10.1021/acs.jmedchem.8b00042

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Article
Discovery of Highly Potent Pinanamine-Based Inhibitors Against
Amantadine- and Oseltamivir-resistant Influenza A Viruses
Xin Zhao, Runfeng Li, Yang Zhou, Mengjie Xiao, Chunlong Ma, Zhongjin Yang, Shaogao Zeng,
Qiuling Du, Chunguang Yang, Haiming Jiang, Yanmei Hu, Kefeng Wang, Chris Ka Pun Mok,
Ping Sun, Jianghong Dong, Wei Cui, Jun Wang, Yaoquan Tu, Zifeng Yang, and Wenhui Hu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00042 • Publication Date (Web): 25 May 2018
Downloaded from http://pubs.acs.org on May 25, 2018
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Discovery of Highly Potent PinanamineꢀBased
Inhibitors Against Amantadineꢀ and Oseltamivirꢀ
resistant Influenza A Viruses
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Xin Zhao†，‖，§，‡，* , Runfeng Li,⊥，‡ Yang Zhou,#，‡ Mengjie Xiao,†，‖，‡ Chunlong Ma,§，Δ
Zhongjin Yang, † Shaogao Zeng, ‖ Qiuling Du, ⊥ Chunguang Yang, ⊥ Haiming Jiang, ⊥
⊥ ,▽
‖
Yanmei Hu,§，Δ Kefeng Wang,† Chris Ka Pun Mok,
Ping Sun,† Jianghong Dong,
Wei Cui, ‖Jun Wang,§，Δ Yaoquan Tu,# Zifeng Yang⊥,*, and Wenhui Hu,†，‖，
*
† State Key Laboratory of Respiratory Disease, Key Laboratory of Molecular Target & Clinical
Pharmacology, School of Pharmaceutical Sciences & The Fifth Affiliated Hospital, Guangzhou
Medical University, Guangzhou 511436, P.R. China
‖ Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou
510530, P.R. China
⊥ State Key Laboratory of Respiratory Disease, National Clinical Research Center for
Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital,
Guangzhou Medical University, Guangzhou 510120, P.R. China
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§ Department of Pharmacology and Toxicology, College of Pharmacy, The University of
Arizona, Tucson, Arizona 85721, United States
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Δ BIO5 Institute, The University of Arizona, Tucson, Arizona 85721, United States
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# Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of
Technology (KTH), AlbaNova University Center, Stockholm SEꢀ100 44, Sweden
▽HKUꢀPasteur Research Pole, School of Public Health, HKU Li Ka Shing Faculty of Medicine,
The University of Hong Kong, 5 Sassoon Road, Hong Kong
ABSTRACT
Influenza pandemic is a constant major threat to public health caused by influenza A viruses
(IAV). IAVs are subꢀcategorized by the surface proteins: hemagglutinin (HA) and neuraminidase
(NA), in which they are both essential targets for drug discovery. While it is of great concern that
NA inhibitor oseltamivir resistant strains are frequently identified from human or avian influenza
virus, structural and functional characterization of influenza HA has raised hopes for new
antiviral therapies. In this study, we explored a structureꢀactivity relationship (SAR) of
pinanamineꢀbased antivirals and discovered a potent inhibitor M090 against amantadineꢀresistant
viruses, including the 2009 H1N1 pandemic strains, and oseltamivirꢀ resistant viruses.
Mechanism of action studies, particularly hemolysis inhibition, indicated that M090 targets
influenza HA and it occupied a highly conserved pocket of the HA2 domain and inhibited virusꢀ
mediated membrane fusion by “locking” the bending state of HA2 during the conformational
rearrangement process. This work provides new binding sites within the HA protein and
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indicates that this pocket may be a promising target for broadꢀspectrum antiꢀinfluenza A drug
design and development.
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INTRODUCTION
Influenza is a severe respiratory disease caused by influenza viruses. The virus belongs to the
Orthomyxoviridae family and is an enveloped negativeꢀsense RNA virus.1 There are three genera
of influenza viruses, A, B and C, but only influenza A virus (IAV) causes pandemics.2 In the last
two decades, we have witnessed the outbreak of influenza epidemics and pandemics, such as
highly pathogenic avian influenza (HPAI), which occurred in 2005 and 2013 with the subtypes
H5N1 and H7N9, respectively.3,4 Furthermore, in the 2009ꢀ2010 influenza season, the H1N1
influenza (swine flu) quickly spread worldwide and caused substantial morbidity and mortality
globally.5 In recent years, another HPAI, H5N6, was identified in the south of China and cases of
human infection were reported.6 Although antiꢀinfluenza vaccines are available, the efficacy is
limited by antigenic drift or shift of virus.7,8 Two classes of antiviral drugs are available on the
market, the neuraminidase (NA) inhibitors (oseltamivir, zanamivir, and peramivir) and Matrix 2
(M2) ion channel inhibitors (amantadine and rimantadine) (Chart S1). An issue facing both
classes of drugs is emerging drug resistance. Nearly all current influenza virus strains are
resistant to amantadine and its derivatives.9ꢀ13 The only available orally administered antiꢀ
influenza drug, oseltamivir, has been documented to have lost its effect in some strains.14ꢀ16
Therefore, discovery of a novel generation of antiꢀinfluenza agents and an understanding of their
molecular mechanisms are urgently needed.
Among the list of drug targets that are currently being pursued in preclinical and clinical
developments, M2 proton channel remains a hot topic. More than 95% of current circulating
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influenza A viruses carry the amantadineꢀresistant AM2ꢀS31N mutation,17 rendering it a highꢀ
profile drug target.18ꢀ28 During the course of developing AM2ꢀS31N inhibitors, it was found that
although some of the compounds inhibit viral replication through AM2ꢀS31N channel blockage,
there are several analogs that inhibit viral replication through AM2ꢀS31Nꢀindependent
mechanisms. Following drug resistance testing selection experiments reveal several mutations in
hemagglutinin (HA),29ꢀ31 indicating HA might be the drug target instead of M2. Nevertheless, the
exact binding site and detailed mechanisms of action of these inhibitors have not yet been fully
explored.
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Small molecules can be used to probe key features of the mechanism of a specific binding site
with a functional protein. Forward chemical genetics operates by using an effective compound to
identify the genetic underpinnings of drug targets.32 Using this approach, in this study, we report
our discovery of a potent antiviral, M090, and its mechanism of action (MOA). Compound
M090 was discovered through a structureꢀactivity relationship (SAR) study (Table 1) of a series
of pinanamineꢀbased antivirals.33ꢀ35 Structural similarity between M090 and reported AM2ꢀS31N
inhibitors led us to hypothesize that M090 inhibits viral replication through AM2ꢀS31N
blockage. However, electrophysiology experiments revealed that M090 did not inhibit the AM2ꢀ
S31N channel. Given the potent antiviral activity and the high selectivity index, we therefore are
interested in applying M090 as a chemical probe to dissect its MOA. Through resistance
selection experiment, we identified a novel binding pocket that is located at HA2. The proposed
MOA was further supported by molecular dynamics simulations and hemolytic fusion assays.
Overall this work suggests a new binding site and the mechanism underlying the interaction of
small molecules with the HA protein of IAV and indicates that this more conserved pocket may
be an advanced target for antiviral drug design and development.
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RESULTS
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Chemistry and SAR study. To study the SAR of pinanamine derivatives using a cytopathic
effect (CPE) assay, we conducted a phenotypic screen for synthetic compounds against the
swineꢀorigin human influenza A/Guangzhou/GIRD/07/2009 (H1N1)36 virus. As shown in
Scheme 1 and Table 1. The synthesis of pinanamineꢀheterocyle derivatives (compounds 1 to 10,
Table 1), 5ꢀposition substituted thiophene linked derivatives (compounds 35 to 41, Table 1) and
compounds 11, 13, 16, 21, 22, 23, 24, 33 and 34 were obtained from commercially available
heterocyclic formaldehyde materials reacting with 3ꢀpinanamine through an easily accessible
reductive amination reaction (Method E).37 For the synthesis of 3ꢀ and 4ꢀ substituted thiophene
derivatives, the routes started from 3ꢀbromothiophene to 3ꢀalkyl substituted thiophene through a
KumadaꢀCorriu coupling reactions (for compounds 19, 20, 31 and 32, Method A),38
subsequently, a Vilsmeier–Haack reaction was conducted to afford corresponding 3ꢀ (Method B) or
4ꢀ (Method C) substituted thiophenecarboxaldehyde (for compounds 12, 14, 15, 25, 26 and 27),39
or 3ꢀ and 4ꢀ substituted thiophenecarboxaldehyde reacted with potassium alkyltrifluoroborate
directly (for compounds 17, 18, 28, 29 and 30, Method D) through a Suzuki−Miyaura crossꢀ
coupling reactions (Method A),40,41 then the same reductive amination reaction gave to the
desired compounds (details were described in the Experimental Section).
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Scheme 1. Synthetic methods of pinanamine derivatives.
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Method D
Method B
S
d
CHO
S
b
CHO
Br
KF3B R
Method A
S
S
R
XMg R
a
d
Br
S
R
OHC
S
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KF3B R
c
OHC
Method C
R
Br
for compounds
19, 20, 31, 32
for compounds
17, 18, 28, 29, 30
for compounds
12, 14, 15, 25, 26, 27
O
H
R'
R'
Method E
NH2
NH
e
Reagents and conditions: (a) Ni(dppp)Cl2, ether, reflux; (b) POCl3, DMF, r.t; (c) nꢀBuLi,
−78°C, THF, then DMF; (d) Pd(dppf)Cl2, K3PO4, H2O/toluene=1:3, 100 °C; (e) NaBH(OAc)3,
CH2Cl2, AcOH, r. t.
Firstly, we explored the SAR of pinanamineꢀheterocyle derivatives (compounds 1 to 10) and
identified compound 7 as the most potent compound in primary CPE assay. These results
indicated that thiophene group may be a prior pharmacophore. We then further obtained several
active compounds through the SAR studies of thiophene based pinanamine derivatives. SAR
exhibited that aliphatic substitutions with middle size in the 3ꢀ or 4ꢀposition (R1 or R2 in Table 1)
of thiophene revealed potent activities (Table 1). The most potent compound with the highest
selectivity index, M090, was selected for followꢀup inhibition studies on multiple strains
(including amantadineꢀresistant and oseltamivirꢀresistant strains) of IAV using CPE (Table 2)
and a plaque reduction assay (PRA) (Figure 1 and S1). Gratifyingly, M090 showed potent
antiviral activities against A/California/07/2009, H1N1 (amantadineꢀresistant, oseltamivirꢀ
sensitive) and A/Texas/04/2009, H1N1 (amantadineꢀresistant, oseltamivirꢀresistant) strains,42
with an PRA EC50 of 0.34 ± 0.016 µM and 0.1 ± 0.009 µM respectively, (Figure 1B and 1C). As
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the structure of M090 fits the pharmacophore model of AM2ꢀS31N inhibitors,26ꢀ28 we
hypothesize that the broadꢀspectrum antiviral activity of M090 might result from its inhibition of
the AM2ꢀS31N proton channel. Therefore, we tested the channel blockage of AM2ꢀS31N by
M090 series in twoꢀelectrode voltage clamp electrophysiological assay (Table S1).43
Surprisingly, M090 showed negligible channel blockage, which suggests its antiviral activity is
independent of AM2ꢀS31N channel blockage. Given the broadꢀspectrum antiviral activity of
M090, we were intrigued to explore its novel MOA.
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Table 1. Inhibitory effect of the synthesized compounds on influenza virus-infected MDCK
cellsa.
Activity
MCC
(ꢁM)c
Compd
.
Structure
(EC50, ꢁM)b
d
−
>100
>100
1
2
−
−
>100
3
60.46 ± 1.75
−
>100
50
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28.60 ± 0.24
50
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6.42 ± 0.18
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−
>100
>100
>100
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9
−
75.00 ± 0.08
10
R1
Me
R2
H
H
H
H
H
H
H
R3
H
H
H
H
H
H
H
−
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25
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Et
−
Br
−
nꢀPr
nꢀBu
OEt
vinyl
−
10.33 ± 2.17
−
1.82 ± 1.04
18
(M090)
H
H
H
H
H
H
H
H
0.30 ± 0.04
25
0.69 ± 0.12
12.5
1.56
12.5
19
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−
−
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H
H
−
12.5
22
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H
H
H
H
H
H
Br
Me
H
H
H
H
H
H
5.16 ± 1.08
6.25
12.5
25
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0.20 ± 0.004
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Et
−
nꢀPr
nꢀBu
vinyl
−
0.46 ± 0.07
−
12.5
12.5
6.25
H
H
H
H
−
25
25
29
30
6.68 ± 0.42
H
H
H
H
H
H
H
H
−
−
−
−
1.56
0.78
25
31
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5
H
H
H
H
H
H
H
H
H
H
Cl
Me
Br
−
−
−
−
−
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0.78
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Et
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0.78
tꢀBu
H
H
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H
H
−
0.78
41
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>100
>100
Amantadine
a Virus strain, A/Guangzhou/GIRD/07/2009 (H1N1).
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b All the compounds were also tested for the inhibitory effect against A/WSN/33 and A/HK/68
strains based on a CCKꢀ8 reagent measurement method44. Compounds 6, 7, 10, 11−18, 23, 24,
26, 28, 30, 36 and 40 exhibited range of micromolar to subꢀmicromolar activity against both
strains (the data were not shown), consistent with this experiment with the Guangzhou strain,
compound 18 (M090) was the most potent inhibitor of the WSN and HK strains too.
c MCC: minimum cytotoxic concentration, or concentration which led to minimal change in
cell morphology after 48 h incubation with compound.
d “−” represent that no viral inhibition occurs when the concentrations of compounds were
lower than MCC.
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Figure 1. Identification of M090 as a potent antiviral against A/California/07/09 and
A/Texas/04/2009. (A) Chemical structure of M090. (B, C) Inhibitory curves and plaques for
M090; The EC50 was analyzed by measurement of percent area of plaques. Each point
corresponds to the mean ± s.d.; data were obtained from two independent experiments.
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Table 2. Effect against a broader panel of influenza A viruses.
Compound
M090
Amantadine Oseltamivir
A/WSN/33
A/PR/8/34
A/PR/8/34
1.48 ± 0.22
2.23 ± 0.60
>100
>100
0.40 ± 0.08
0.13 ± 0.03
H1N1
6.85 ± 1.85
>100
>10
(NAꢀH274Y)
Antiviral
activity
A/HK/68
5.38 ± 0.32
4.55 ± 0.80
1.00 ± 0.50
NDb
2.30 ± 0.90
0.99 ± 0.05
H3N2
(EC50, µM)a
A/Aichi/2/68
A/duck/Guangd
ong/1/1996
H7N3
H9N2
4.58 ± 0.66
6.79 ± 0.44
ND
ND
2.67 ± 0.56
>10
A/duck/HK/
Y280/97
a
The antiꢀinfluenza activity of M090 was evaluated by CPE assay and validated by PRA assay
(Figure S1).
b ND, not detected.
Mechanism of Action Study. The replication cycle of the influenza virus begins at absorption
to and ends at the production of its progeny viruses; the whole cycle requires approximately 8 to
10 h. There are three steps, relative to virus entry at time 0, in this process: the early stage of
attachment and entry (ꢀ1 to 2 h), the middle stage of endosomal release, genome replication and
viral protein translation (2 to 8 h), and the late stage of virion assemble and release of progeny
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viruses (8 to 10 h).45 Defining ꢀ1 h as the time point of absorption, an unbiased drug timeꢀofꢀ
addition (TOA) experiment46 was performed.
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The whole life cycle of virus was divided into eight treatment intervals of 1 to 2 h each
(Figure 2A); M090 was administered for treatment, and the yield was measured using TCID50
(virus infectious dose that can produce pathological change in 50% of cell cultures inoculated.)
testing. The intervals of ꢀ1 to 2 h and 0 to 2 h postꢀvirus absorption showed inhibition due to the
presence of M090 (Figure 2B). This finding indicates that M090 blocked the early steps of the
virus life cycle, as well as the entry stage, which includes attachment, endocytosis and fusion.47
The attachment, endocytosis and fusion process is related to two viral proteins of IAV, M2 and
HA.8,48 As M090 did not inhibit the AM2ꢀS31N proton channel as shown by the TEVC assay
results, we hypothesize that the HA protein may be the target of this series of antiviral
compounds such as M090. HA is encoded by the fourth viral RNA segment, and its precursor,
HA0, undergoes extraꢀ or intracellular cleavage into two subunits, HA1 and HA2.49,50 In the virus
entry progress, HA1 regulates virus attachment to the sialic acid (SA) receptor of the host cell,
which appears as hemagglutination.51,52 As shown in hemagglutination inhibition (HI) assays,
M090 did not inhibit the virusꢀinduced aggregation of chicken erythrocytes (Figure S2),
revealing that there was no interaction between M090 and the HA1 domain.
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Figure 2. Time-of-addition approach to identify the targeted stage of M090. (A) Time course
design. MDCK cells were infected with H1N1 (A/PR/8/34) at 100 TCID50. The virus absorption
was indicated as ꢀ1 h and M090 was added at ꢀ3 to ꢀ1, ꢀ1 to 0, ꢀ1 to 2, 0 to 2, 2 to 4, 4 to 6, 6 to 8,
8 to 10 h postꢀinfection. After each treatment period, M090 was removed and cells were
incubated with culture medium till the end point. (B) M090 was added at each interval, and the
virus yield was determined by measuring the TCID50. *p<0.05, ** p<0.01, compared to the
condition without drug treatment.
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Identification of Binding Sites by Drug Resistance Selection Experiments. To identify the
binding site of M090, resistanceꢀinduction experiments were performed against the
A/Guangzhou/GIRD/07/2009 (H1N1) virus in MDCK cells (Table 3). At each passage, serial 2ꢀ
fold dilutions of M090 were selected (43 ~ 0.042 ꢁM) and virus that developed 50% CPE was
picked for the subsequent passage. At the first two passages, the virus progeny maintained full
drug sensitivity. Drug resistance developed from passage 3, and the EC50 increased threeꢀfold.
Resistance gradually increased in passages 4 to 6 and became significant after passage 6. After
passage 7, the virus was fully resistant to M090.
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Table 3. Resistance Development under Pressure of M090a.
Passage no.
EC50 (µM)b
0.30
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0.34
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1.70
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25.6
>43.0
>43.0
>43.0
Resistance to M090 developed from passage 3 and became significant after passage 6.
a The parent strain is A/Guangzhou/GIRD/07/2009.
b EC50 values tested after each passage.
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We sequenced the M and HA RNA segments of the resistant strain of passage 8 and compared
the sequences with those of the parent A/Guangzhou/GIRD/07/2009 virus, which was reported in
the Influenza Research Database.53 Two substitutions were found (changes in basic groups are
shown in Figure S3). Both substitutions occurred in the HA protein, one is A15T, which located
at the signal peptide of HA50 and does not exist in the mature virus protein. Logically, this
substitution could not emerge from drug pressure. The other site, HAꢀE418D, known as HA2ꢀ
E74D, is located on the top of the αꢀhelix of the HA2 trimer (Figure 3). This mutant is the sole
reasonable active site of M090.
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Figure 3. Crystal structure (PDB entry: 3AL4) and M090-induced substitution of HA. HA
structure from the swineꢀorigin A (H1N1)ꢀ2009 influenza A virus A/California/04/2009.54 The
RNA sequences of this strain are identical to those of A/California/07/2009 and
A/Guangzhou/GIRD/07/2009 that we have used. (A) Aminoꢀacid substitution found in HA. (B,
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C) Top and side view of the HA model. The M090ꢀresistant site HA2ꢀE74 is highlighted by
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spherical models in red and blue, respectively.
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Molecular Dynamics (MD) Simulations. The potential binding sites of M090 (Figure 4)
were characterized using SiteMap,55 and M090 was docked into these sites using Glide.56 The
topꢀranked binding poses were clustered. The most favorable docking score in each cluster
(Figure S4) was chosen and further refined by MD simulations.
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Figure 4. Potential binding sites of M090 in wild type HA protein (PDB entry: 3AL4). (A)
Front and top views of potential binding sites (highlighted in red), HA2ꢀE74 is highlighted by
stick model in green. (B) 2D model of the HA binding pocket suggested by docking.
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Figure 5. Predicted M090-HA2 structure. (A) Adjacent to HA2ꢀ74, M090 fits well in the pocket
between a long αꢀhelical segment and a loop of the HA2 monomer. (B) Binding mode of M090
with protein. (C) Time evolution of the RMSD values of M090 and HA protein during the MD
simulation.
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The stability of the predicted conformations of the M090ꢀHA2 complex were evaluated by the
average rootꢀmeanꢀsquare deviation (RMSD) value of HA protein and M090. As shown in
Figure 5, in the HA2 monomer, a short helix and a long helix are linked by a loop (Figure 5A).
The predicted binding pose of M090 in the binding pocket is located at the interface of the long
helix (residues 82 to 93) and loop (residues 57 to 69) (numbering started from the HA2 segment).
M090 fits well in the pocket. Its pinane scaffold forms hydrophobic interactions with HA2ꢀAla65
and HA2ꢀ Val66, the thiophene ring lays in the hydrophobic region formed by HA2ꢀIle89,
Tyr312, and HA2ꢀTrp92, the cyclopropyl group forms hydrophobic interactions with HA2ꢀPhe88
and Pro303, and the hydrogen bond between NꢀH and Tyr305 enhances the binding of the
compound with the protein (Figure 5B).
During low pHꢀtriggered membraneꢀfusion, the conformation of the HA2 monomer changes
from a folding shape to a needle shape, and HA2 attaches to the host cell membrane (Figure
S5)57. Around the experimentally observed M090-induced mutant site HA2ꢀE74, Glu74 directly
interacts with the adjacent Arg76 residues to form three hydrogen bonds to stabilize the bent
conformation of HA2 (Figure 6A). MD simulations indicated that in the presence of M090, site
1, which is close to the binding pocket, has higher Hꢀbond occupation than the other two binding
sites (Figure 6B). Stabilized by M090, the initial HA2 bending state is “locked”, and the HA2
monomer is accordingly blocked to from forming a needleꢀshaped conformation, which helps the
protein attach to the host cell. The in silico results are consistent with the experimental drugꢀ
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resistant mutation E74D, which indicates that the Glu74 mutant is a “shorter form”, namely, Asp,
and the hydrogen bond interaction is weakened and breaks down easily despite drug binding.
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Figure 6. Mode of M090 action studied by MD simulation. (A) M090 blocks the
conformational change of the HA2 monomer by enhancing the Hꢀbond interaction between the
long helix and the loop. (B) Time evolution of the number of Hꢀbonds of each site during the
MD simulation.
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Hemolytic Fusion Assays. MD simulations demonstrated that M090 inhibits the membraneꢀ
fusion process by blocking the conformational changes of HA2; thus, the HAꢀmediated
hemolysis inhibitory effect of M090 was tested for validation.
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The optical density (OD540) value was measured after the chicken red blood cells (CRBC)
were mixed with the virus together with different concentrations of M090. Two different viral
strains (H1N1, A/PR/8/34 and H3N2, A/Aichi/2/68, which represent the two HA phylogenetic
groups respectively) were used to induce hemolysis. A known hemolysis inhibitor, Arbidol58,
was used as our control. As shown in Figure 7, the hemolysis inhibition was occurred in doseꢀ
dependent manner using the two virus strains.
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Figure 7. Hemolysis inhibition measurements. (A) H1N1, A/PR/8/34. (B) H3N2,
A/Aichi/2/68. The level of hemolysis inhibition was measured at different concentration of
M090. At each concentration, the inhibition effect of M090 (blue curves) was more potent than
the positive control, Arbidol (red curves). Each point corresponds to the mean ± s.d.; The results
were repeated in two independent experiments.
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DISCUSSION
Based on the differences in nucleotide sequences, the HA subtypes are divided into two
phylogenetic groups, group 1, including H1, H5 and H9, and group 2, including H3 and H7.59
Many fusion inhibitors were reported in literature, lots of them were HA group 1 specific, such
as BMS-19994560, RO5464466 series,61,62 CL-385319 series63ꢀ65 and so on.66ꢀ71 Recently, a
potent peptidic inhibitor of HA was identified, it was also group 1 specific.72 In the case of group
2, THBQ and its analogues73ꢀ75 and a series of azaspiro derivatives76 were identified to target H3
subtype. Another broadꢀspectrum antiviral drug, Arbidol, was characterized as a HA2 inhibitor.58
The coꢀcrystallization of it with HA was analyzed and revealed that its binding site was similar
with THBQ.77 We aligned the sequences which cover the potential binding pocket of M090 from
two sets of HA subtype strains, set 1 was comprised of five H1, one H5, one H9 strains, and set 2
was comprised of two H3, two H7 strains (Figure S6). It was found that the sequence similarity
of the strains from group 1 was more than 90 % while the strains from group 2 has more than
50% similarity. Remarkably, the escape mutation site, E418, is highly conserved among the two
sets. Comparing to the antiviral effect of M090 on different virus strains including H1 and H9
(belong to group 1) as well as, H3 and H7 (belong to group 2) (Table 2), we found that this
region is not absolutely group specific and may be used to design advanced inhibitors that target
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both phylogenetic groups of HAs and potentially explore broadꢀspectrum antiviral drugs. At the
same time of preparing this paper, a series of anilineꢀbased HA inhibitors were published and
reported their antiviral activities against H1 and H5 HAs, the MD simulation study demonstrate
that the binding site of these inhibitors overlaps with the binding cavity of THBQ,78 it may also
inhibit the group 2 HA viruses. That work partially support our research strategy and
demonstrated the rationality of the new binding site we found. These results can serve as a
starting point for our work in coming future including the further optimization of M090 and its
coꢀcrystal complex with HA aiming for the analysis of binding site confirmation.
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CONCLUSION
Influenza always threatens causes potential new pandemics and annual epidemics; thus, new
therapeutics, especially with new targets and mechanisms of action, constitute a crucial strategy
to combat these outbreaks. The process of HA growth and the function of HA offers targets for
therapeutic intervention.79 Here, using a forward chemical genetics approach, we identified a
potent antiviral agent, M090, and its binding site in the HA2 subunit. Extensive MOA studies of
M090 indicated that small molecules can bind to a highly conserved segment of HA2, a new
binding site located in the space of the long helix and the loop of the HA2 monomer. In the
presence of inhibitor, low pHꢀinduced membrane fusion was blocked via conformational
rearrangements that led to inhibition of HA2.
This work identifies highly potent inhibitors against amantadineꢀ and oseltamivirꢀresistant
influenza A viruses and provides new insights into the specific binding site of small molecules
with HA protein and implications regarding structureꢀbased rational drug design. M090 and its
furtherꢀmodified compounds potentially could be components in combination therapy with
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approved antiꢀinfluenza drugs to overcome highly pathogenic virus strains. Indeed, this study
proposed a promising binding site for the design and development of broadꢀspectrum antiviral
drugs, and these small molecules may have a more resistant genetic barrier under drug pressure
due to the conservation of the binding site. In addition, HA contributes to the early stage of the
viral cycle, and newly designed HA inhibitors based on this binding site can be used for their
prophylactic and therapeutic efficacy.
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EXPERIMENTAL SECTION
Chemistry. All commercially available compounds and solvents were reagent grade and were
used without further treatment unless otherwise noted. Reactions were monitored by TLC using
Qing Dao Hai Yang GF254 silica gel plates (5 x 10 cm); zones were detected visually under
ultraviolet irradiation (254 nm) by either spraying with an ethanol solution of ninhydrin or by
treatment with iodine gas. Compounds were purified by silica gel column chromatography
1
performed on silica gel (200 to 300 mesh) from Qing Dao Hai Yang and characterized by H
13
NMR, C NMR and ESIꢀMS. The NMR spectra were recorded on a Bruker NMR AVANCE
400 (400 MHz) or a Bruker NMR AVANCE 500 (500 MHz), and the NMR reagents CDCl3 and
DMSOꢀd6 were used as internal standards. Chemical shifts (δ) were recorded in parts per million
and coupling constants (J) in hertz (Hz). MS data were measured on an Agilent MSDꢀ1200 ESIꢀ
MS system. The purity of compounds was analyzed by HPLC performed on an Agilent Sunfire
C18 (150×4.6mm, 3.5µm) column, solvent, H2O (0.1 % HCOOH), 0.8 mL/min flow rate or 16
min gradient, 10 % to 100 % TFA in H2O (10 % to 80% KH2PO4 in H2O for compounds 25 and
37), 1.0 mL/min flow rate, the peak was detected at 254 nm. All compounds submitted for
testing in biological assays were confirmed to be >95% purity by HPLC. Detailed regarding
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inhibitor characterization are provided in the Supporting Information. For use in assays,
compounds were prepared as hydrochlorides or phosphates and dissolved in DMSO.
Detailed synthesis procedures.
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Method A. To a solution of Ni(dppp)Cl2 (1 mol%) in anhydrous ether (20 mL), in the N2
atmosphere, Cyclopentylmagnesium chloride or Cyclohexylmagnesium chloride (2M in ether, 30
mmol) was added dropwise at 0 °C, then the 3ꢀBromothiophene (25 mmol) in ether was added
dropwise. The resulting solution was refluxed for 15h, then cooled to r. t. and quenched by 2M
HCl. The mixture was extracted using ethyl acetate (100 mL×3). The organic layer was washed
with brine, dried with Na2SO4 and the solvents were removed. The residue was purified by silica
gel column chromatography to afford the corresponding alkylthiophene.
Method B. To a solution of DMF (5 mL), POCl3 (15 mmol) was added, the mixture was
stirred for 1 h at r. t. Then the alkylthiophene (10 mmol) in DMF (5 mL) was added dropwise
and stirred for 1 h at 100 °C. The solution was then cooled to 0 °C and quenched by saturated
NaHCO3 solution. The mixture was extracted by ethyl acetate (3 x 20 mL), the organic layer was
washed with brine, dried with Na2SO4 and the solvents were removed. The residue was purified
by silica gel column chromatography to afford the corresponding aldehyde.
Method C. To a solution of alkylthiophene (10 mmol) in anhydrous ether (10 mL), nꢀBuLi
(2M in ether, 15 mmol) was added dropwise at 0 °C in the N2 atmosphere. The mixture was
refluxed for 1 h. The solution was then cooled to r. t. and DMF (1 mL) was added. The resulting
solution was stirred for 2 hs and quenched by saturated NH4Cl solution. The mixture was
extracted by ethyl acetate (3 x 20 mL), the organic layer was washed with brine, dried with
Na2SO4 and the solvents were removed. The residue was purified by silica gel column
chromatography to afford the corresponding aldehyde.
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Method D. To a solution of 3ꢀ or 4ꢀ substituted thiophenecarboxaldehyde (35 mmol), K3PO4
(105 mmol), Pd(dppf)Cl2.CH2Cl2 (0.7 mmol) and in toluene/H2O = 3/1 (140 mL), Potassium
Alkyltrifluoroborate (42mmol) was added in the N2 atmosphere. The resulting solution was
refluxed for 10 h. Then 50 mL water was added. Subsequently, the mixture was extracted using
ethyl acetate (100 mL×3). The organic layer was washed with brine, dried with Na2SO4 and the
solvents were removed. The residue was purified by silica gel column chromatography to afford
the corresponding alkylthiophene.
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Method E. To a solution of (1R,2R,3R,5S)ꢀ(−)ꢀisopinocampheylamine (6.5 mmol) in CH2Cl2
(20 mL), aldehyde (1.5 equiv., 9.8 mmol) was added. The reaction mixture was stirred at r.t. for
1 h, followed by the addition of sodium triacetoxyborohydride (26 mmol). The reaction was then
stirred for 10 h and quenched by the addition of H2O and extracted with CH2Cl2 (3x20 mL). The
combined organic layers were washed with brine, dried with Na2SO4 and evaporated to dryness.
The residue was purified by silica gel column chromatography, treated with saturated
HCl/CH3OH (20 mL), then evaporated to dryness. The insoluble material was washed with
diethyl ether (3x20 mL) to afford the secondary amines as the corresponding hydrochloride salts.
Cytopathic Effect (CPE) Assay. In the CPE assay, MDCK cells were grown to a confluent
monolayer in a 96ꢀwell culture plate at a concentration of 5×104/well for 24 h. The medium was
removed, and the cells were rinsed twice. An infectious virus at 100 TCID50 (the influenza virus
infectious dose that can produce pathological change in 50% of cells during 48 hours inoculated
time period) was inoculated into the MDCK cells, which were then incubated for 2 h at 37 °C in
5% CO2. The virus supernatant was removed, which was followed by the addition of serial twoꢀ
fold dilutions of antiviral compounds in DMEM containing 1.5 µg/mL trypsin. After being
incubated at 34 °C in 5% CO2 for 48 h, the infected cells displayed 100% CPE under the
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microscope, and the CPE percentages in the antiviral compoundꢀtreated groups were recorded.
The EC50 values were calculated using a nonꢀlinear regression model in GraphPad Prism.
Cytotoxicity Assay. The cytotoxicity assay was carried out using similar method as the CPE
assay. MDCK cells were grown to a monolayer in a 96ꢀwell culture plate at a concentration of
5×104/well. After 24 hours, the medium was removed, and the cells were rinsed with PBS twice.
Compounds with twoꢀfold serial dilution of compounds in DMEM were added into the cells.
After incubation for 48 hours, the cell survival rate of each well was observed under the
microscope and the concentration which led to minimal change for cell morphology was counted
as the minimum cytotoxic concentration.
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Plaque Reduction Assay. A monolayer of A549 cells was infected with 0.01 MOI influenza
A viruses for 1 h at 37 °C. The inocula were removed, and the cells were washed twice with
phosphateꢀbuffered saline (PBS). The cells were overlaid with 1% agar DMEM with amantadine
or one of the synthesized compounds in the presence of 2 ꢁg/mL trypsin and 0.3% BSA. Two to
three days after infection, the monolayers were fixed and stained with 0.1% crystal violet
solution. The viruses used for this assay were A/California/07/2009 (H1N1) and
A/Texas/04/2009 (H1N1). To use the virus strains A/WSN/33 (H1N1), A/HK/68 (H3N2) and
A/PR/8/34 (H1N1), MDCK cells were used instead of A549 cells.
Time-of-addition approach. MDCK cells were grown to a confluent monolayer in a 24ꢀwell
culture plate and washed twice with PBS. 100 TCID50 of influenza A virus (A/PR/8/34) at was
inoculated into the MDCK cells (0.5 mL per well) and allowed to absorb at 4 °C for 1 h. The
wells were then washed with PBS. The plates received medium containing 1.5 ꢁg/mL TPCKꢀ
treated trypsin (0.5 mL per well) and were incubated in a CO2 incubator at 37 °C for 10 h.
Compound (10 ꢁM) was added at ꢀ3 to ꢀ1, ꢀ1 to 0, ꢀ1 to 2, 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10 h
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postꢀinfection. After each treatment period, the monolayer was washed of drug with PBS, and
medium containing 1.5 ꢁg/mL TPCK (0.5 mL per well) was added. After 24 h, the plate was
frozen and thawed, the supernatant was collected, and TCID50 was measured to determine the
virus yield.
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Two-Electrode Voltage Clamp (TEVC) Assay.80 Selected compounds were tested in a
TEVC assay using Xenopus laevis frog oocytes microinjected with RNA expressing the wild
type or S31N mutant of the A/M2 protein. The potency of the inhibitors was expressed as
percentage inhibition of A/M2 current, as observed after 2 min of incubation with 100 ꢁM
compounds at pH 5.5. All measurements were repeated two times with different cells.
Hemagglutination inhibition (HI) assay. Compound from a serial twoꢀfold dilution in PBS
was mixed with an equal volume of influenza virus (A/PR/8/34). After a 1ꢀh incubation in a Vꢀ
bottom 96ꢀwell plate, 50 ꢁL of compoundꢀvirus preparation was added to an equal volume of
0.5% freshly prepared chicken red blood cells in Alsever's solution. The mixture was incubated
for 40 min at room temperature before observing erythrocyte aggregation on the plate.
Serial passage experiments and resistant mutation identification. MDCK cells were
infected with influenza H1N1 virus (A/Guangzhou/GIRD07/2009) at 100 TCID50 for 2 h; the
inoculum was removed, and cells were incubated with M090 at the concentrations of twoꢀfold
serial dilutions beginning at MCC or in the absence of M090 for 2 days to evaluate drug
pressureꢀselected mutations in vitro. At the endpoint of each passage, the viruses that developed
a significant 50% CPE were harvested and subjected to the following passages. The titer sample
of harvested virus at the 8th passage was subjected to sequencing. Influenza viral RNA was
isolated using a QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany). The eight segment
cDNAs were generated with reverseꢀtranscription polymerase chain reaction (PCR) using
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specific primers (Table S2). Reverseꢀtranscription PCR products were identified by agarose gel
electrophoresis. PCR products were sequenced by BGI, Inc. (Shenzhen, CHN).
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Docking and molecular dynamics (MD) simulations. The crystal structure of swineꢀorigin A
(H1N1)ꢀ2009 influenza A virus HA (PDB: 3AL4) was obtained from the Protein Data Bank
(http://www.rcsb.org/pdb/) and prepared using the Protein Preparation Wizard.81 The missing
sidechains were added, and the protonation states were determined using PROPKA. The
structure thus obtained further underwent a restrained energy minimization. The compound was
prepared using LigPrep (Schrödinger, LLC), and 30 conformations were generated for docking.
The binding sites were characterized using SiteMap,55 and all conformations were docked into
the active site using Glide.56
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MD simulations were performed using the GROMACS code.82 The Amber99SBꢀildn force
field83 was used for the protein, and the GAFF force field84 was used for the compound. Each
HA/compound complex was solvated in a 15*15*15 nm3 cubic box with 144538 TIP3P85 water
molecules. To neutralize the system and to prepare a final NaCl concentration of 0.15 M, 304
Na+ and 296 Clꢀ ions were added. The system was energy minimized with the steepest descent
algorithm for 1000 steps. Thereafter, 200ꢀps isothermalꢀisochoric (NVT, with T=300 K) and
200ꢀps isothermalꢀisobaric (NPT, with T=300 K and P=1 atm) MD simulations were performed
to equilibrate the system. The harmonic restraint with a force constant of 1000 kJ molꢀ1 nmꢀ2 was
used during the simulations. The equilibrated system was then subjected a simulation of 150 ns
without any restraint. During the equilibration and production runs, the time step was 2 fs, and
the LINCS algorithm86 was applied to constrain the bonds involving hydrogens. The van der
Waals and shortꢀrange electrostatic interactions cutoff was set to 10 Å, and the particleꢀmesh
Ewald (PME) method87 was used to treat longꢀrange electrostatics.
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Hemolysis inhibition assay. A 100ꢀµL influenza virus (A/PR/8/34) or (A/Aichi/2/68) sample
was incubated with serial twoꢀfold dilution of compounds in PBS for 30 minutes at 37 °C, and an
equal volume of chicken red blood cells with citric acidꢀsodium citrate (pH 5.0) was added. After
incubation at 37 °C for 30 minutes, the mixture was centrifuged (400×g, 8 min, 4 °C). Then, 300
µL of the supernatant was transferred to a 96ꢀwell plate, and the absorbance was measured at 540
nm with a Thermo Multiskan MK3 Spectrum spectrophotometer. The value of the mockꢀinfected
samples served as the background. IC50 was expressed as the compound concentration
that caused 50% inhibition of hemolysis.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications website
Details regarding inhibitor synthesis, characterization and 1H NMR and 13CNMR spectra of
new compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Email: zhaoxin2017@foxmail.com (X. Z.)
*Email: jeffyah@163.com (Z. Y.)
*Email: huwenhui@gzhmu.edu.cn (W. H.)
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