Development of novel anti-influenza thiazolides with relatively broad-spectrum antiviral potentials

Article abstract of DOI:10.1128/AAC.00222-20

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug resistance to specific anti-influenza virus drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity

Full text of DOI:10.1128/AAC.00222-20

AAC Accepted Manuscript Posted Online 20 April 2020
Antimicrob. Agents Chemother. doi:10.1128/AAC.00222-20
Copyright © 2020 American Society for Microbiology. All Rights Reserved.
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Development of Novel Anti-influenza Thiazolides with
Relatively Broad-spectrum Antiviral Potentials
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Lei Zhao , Yunzheng Yan , Qingsong Dai , Xingzhou Li , Ke Xu , Gang Zou ,
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Keyu Yang , Wei Li , Xiaojia Guo , Jingjing Yang , Yuexiang Li , Qing Xia ,
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Ruiyuan Cao *, Wu Zhong *
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National Engineering Research Center for the Emergency Drug, Beijing
Institute of Pharmacology and Toxicology, Beijing 100850, P.R. China
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State Key Laboratory of Virology, College of Life Sciences, Wuhan University,
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Wuhan 430072, P.R. China.
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Institut Pasteur of Shanghai, Chinese Academy of Sciences, 320 YueYang
Road, Shanghai 200031, China
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State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing
100191, China.
* Corresponding author: zhongwu@bmi.ac.cn, 21cc@163.com
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Abstract
Seasonal and pandemic influenza causes 650,000 deaths annually in the
world. The emergence of drug-resistance to specific anti-influenza drugs such
as oseltamivir and baloxavir marboxil highlights the urgency of novel
anti-influenza chemical entity discovery. In this study, we report a series of
novel thiazolides derived from an FDA-approved drug nitazoxanide with
antiviral activity against influenza and a broad range of viruses. The preferred
candidates 4a and 4d showed significantly enhanced anti-influenza potentials
with 10-fold improvement, compared with nitazoxanide, and were effective
against a variety of influenza subtypes including oseltamivir-resistant strains.
Notably, the combination using of compounds 4a/4d and oseltamivir
carboxylate or zanamivir displayed synergistic antiviral effect against
oseltamivir-resistant strain. Mode of action analysis demonstrated that
compounds 4a/4d acted at the late phase of viral infection cycle through
inhibiting viral RNA transcription and replication. Further experiments showed
that treatment with compounds 4a/4d significantly inhibited influenza virus
infection in human lung organoids, suggesting the druggability of the novel
thiazolides. In-depth transcriptome analysis revealed a series of up-regulated
cellular genes that may contribute to the antiviral activities of 4a/4d. Together,
our study pointed the optimization direction of nitazoxanide as anti-influenza
drug, and discovered two novel-structured candidates 4a/4d with relatively
broad-spectrum antiviral potential.
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Keywords: influenza virus, thiazolides, antiviral, synergetic effects
Introduction
Seasonal influenza causes heavy burden to public health annually, and
pandemic influenza remains one of the most concerned medical issues in the
world (1, 2). Vaccine is the most effective means to prevent influenza, and
antiviral therapy is essential for clinical treatment of influenza. Currently, three
types of influenza vaccines are clinically available: trivalent/quadrivalent
inactivated vaccines, live attenuated vaccines and the recombinant vaccine
Flublok (3). Antiviral drugs including neuraminidase (NA) inhibitor (NAI)
oseltamivir, M2 ion channel inhibitor amantadine and polymerase acidic protein
inhibitor baloxavir marboxil played important roles in the combat against
influenza. As a novel cap-dependent endonuclease inhibitor, baloxavir
marboxil has been approved in Japan and United States for treatment of
influenza virus infection by the U.S. Food and Drug Administration (FDA) (4).
Notably, a single oral dose could achieve of acute uncomplicated influenza.
However, the emergence of drug resistance is rendering ineffective of current
drugs. The Centers for Disease Control and Prevention (CDC) recommended
discontinued use of M2 channel blockers (amantadine and rimantadine) (5).
The emergence of oseltamivir-resistance and baloxavir-resistance strains is
also worth noticing (6-7).
Nitazoxanide (NTZ) is an FDA-approved antiprotozoal thiazolide drug whose
broad-spectrum antiviral potential was highlighted in recent years (8). NTZ is
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effective to arboviruses including Zika virus (9), Japanese Encephalitis virus
(10) and Chikungunya virus (11). Studies suggested the possible mechanisms
underlying the antiviral activity of NTZ: it induces transcription of the antiviral
phosphatase GADD34 and enhances the activities of several antiviral factors
including RIG-I-like receptor, interferon regulatory factor 3, interferon and
mitochondrial antiviral signaling protein (12). For inhibition of important
hepatitis virus, NTZ was reported effective against hepatitis B virus (HBV) and
hepatitis C virus (HCV) as well (13, 14). The clinical trial revealed that
combination of nitazoxanide, peginterferon alfa-2a, and ribavirin increased the
percentages of patients with rapid and sustained virologic responses.
Thiazolides was found to elicit host anti-viral innate immunity and reduce HIV
replication (15). NTZ exhibited in vitro activity against Middle East Respiratory
Syndrome Coronavirus (MERS-CoV) and other coronaviruses by inhibiting
expression of the viral N protein (16). For treating of viral diarrhea, clinical
studies suggested that NTZ may play important roles in managing adult viral
gastroenteritis caused by Norovirus (17) and children viral diarrhea caused by
rotaviruses (18). In addition, NTZ was reported to have antiviral effect against
rubella virus and vaccinia virus (19, 20).
Researchers have repurposed NTZ as a new generation anti-influenza drug.
NTZ inhibits influenza replication by targeting the maturation of viral
hemagglutinin (HA) (21). Moreover, NTZ showed potent antiviral activity for
drug-resistant strains of seasonal
A
(H3N2) viruses carrying NAI
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resistance-associated NA substitution (22). NTZ was shown to inhibit
replication of influenza A (H1N1) pdm09 and B viruses in vitro (23). For avian
influenza A (H5N9) and canine influenza A (H3N8) viruses, NTZ was reported
to inhibit these non-human influenza viruses potently (24, 25). A clinical trial
focusing on the treatment of influenza using NTZ showed that treatment with
nitazoxanide 600 mg twice daily for 5 consecutive days was associated with a
reduction of the duration of symptoms in patients with acute uncomplicated
influenza (26). However, as revealed by a recent clinical study, NTZ failed to
meet clinical endpoints in treatment of respiratory illness, and improvement
was needed to enhance the antiviral efficacy of this drug (27). In this study, we
made structural modifications of tizoxanide, the bioactive form of NTZ, to
generate a series of novel thiazolides. We attempt to obtain the
next-generation antiviral thiazolides candidates with enhanced anti-influenza
potentials. The candidates 4a and 4d exhibited significantly enhanced
anti-influenza potentials compared with tizoxanide, and had significant
synergistic antiviral effect when used together with other first-line anti-influenza
drugs such as oseltamivir and zanamivir. Mechanism of action analysis
revealed that 4a/4d modulated intracellular events of influenza virus infection,
and the up-regulated cellular genes obtained through in depth transcriptome
analysis give the clue of the specific antiviral target of 4a/4d. Antiviral effects of
4a/4d were validated using a lung organoid infection system, suggesting the
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clinical perspective of these preferred compounds.
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Materials and Methods
Cell Lines and Virus Infection
Madin-Darby canine kidney (MDCK), human embryonic kidney (HEK) 293T,
and human alveolar type II-like epithelial (A549) cells were purchased from the
American Type Culture Collection (ATCC, Manassas, VA) and maintained at
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37℃ and 5% CO in Dulbecco’s modified Eagle’s medium (DMEM, Gibco)
supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL of
penicillin, and 100 μg/mL of streptomycin.
Influenza A/Puerto Rico/8/1934 (H1N1) (PR/8), A/California/07/2009
(H1N1)pdm09 (CA/07), A/LiaoNing-ZhenXing/1109/2010 (H1N1, natural
oseltamivir-resistant isolate) (ZX/1109), A/TianJin-JinNan/15/2009 (H1N1,
natural oseltamivir-resistant isolate) (JN/15), PR/8-R292K mutant (H1N1,
recombinant
oseltamivir-resistant
strain),
(H7N9,
and
low-pathogenicity
were
A/Guangdong/17SF003/2016-NIBRG-375
GD/17SF003)
propagated for 48h at 37℃ in the chorioallantoic cavity of 8~10-day-old
embryonated chicken eggs. Influenza A/WSN/33 (H1N1), A/Hongkong/8/68
(H3N2)
(HK/68)
and
influenza
B/Xiamen/N912/2014(Yamagata),
B/Xiamen/N843/2014(Victoria) were propagated in MDCK cells for 3 days at
33 to 37℃ in serum-free Dulbecco’s modified Eagle’s medium/Ham’s F-12
medium (DF-12) containing 2μg/mL tosylamido phenylethyl chloromethyl
ketone (TPCK)-treated trypsin (Sigma). Oseltamivir-resistant strain (natural
isolate ZX/1109), B/Xiamen/N912/2014 and B/Xiamen/N843/2014 were kindly
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provided by Dr. Yuhuan Li (Institute of Medicinal Biotechnology, CAMS) and Dr.
Yixin Chen (School of Public Health, XIAMEN University), respectively. The
other virus strains were stored in our lab. Virus yield was determined by plaque
assay, HA titration or 50% tissue culture infective dose (TCID50) infectivity
assay.
Compounds
Synthesis of ethyl(2-((5-nitrothiazol-2-yl) carbamoyl)phenyl) carbonate
(4a). Tizoxanide (1.00 g, 3.77 mmol) were added to anhydrous dimethyl
formamide (5 mL), anhydrous pyridine (2.5 mL) and acetonitrile (1.5 mL) at
2 ℃ ~ 5 ℃, then ethyl chloroformate was added drop by drop. The mixture
was stirred at room temperature for 6 h, poured into water and then extracted
by ethyl acetate. The resulting precipitate was filtered, washed with water,
saturated salt water in sequence, followed by drying with anhydrous sodium
sulfate. The product was purified by flash chromatography on silica gel. Yield,
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83.5%; m.p.:156-158℃. H-NMR(CDCl
3
, 400 MHz)δppm: 11.73 (s, 1H), 8.23
(s, 1H), 8.11 (dd, J = 1.68, 7.84 Hz, 1H), 7.72 - 7.47 (m, 2H), 4.39 - 4.36 (m,
+
2H), 1.41 (t, J = 7.14 Hz, 3H). HRMS (ESI+) m/z (M+H) calculated for
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C H N O S: 338.0369; found: 338.0442.
Synthesis of 2-((5-nitrothiazol-2-yl)carbamoyl)phenyl butane-1-sulfonate
(4d). Tizoxanide (0.50 g, 1.89 mmol) was stirred at room temperature in
anhydrous tetrahydrofuran (20 mL) until dissolved completely. A mixture of
triethanolamine (0.38 g, 3.78 mmol) and butanesulfonyl chloride (0.59 g, 3.77
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mmol) was added to the solution and stirred at room temperature. The mixture
was poured into water and extracted by ethyl acetate. The resulting precipitate
was filtered, washed with water, saturated salt water in sequence, followed by
drying with anhydrous sodium sulfate. The product was purified by flash
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chromatography on silica gel. Yield, 92.0%; m.p.:142-144℃; H-NMR(CDCl
3
,
400 MHz) δ ppm:10.81 (s, 1H), 8.25 (s, 1H), 8.01 (dd, J = 1.82, 7.70 Hz, 1H),
7.72 - 7.55 (m, 1H), 7.53 - 7.50 (m, 2H), 3.47(t, J = 8.0 Hz, 2H), 2.04 - 2.00 (m,
+
2H), 1.57 - 1.51 (m, 2H), 0.97 (t, J = 8.0 Hz, 3H); HRMS (ESI+) m/z (M+H)
calculated for C14
carboxylate, Peramivir Trihydrate, Zanamivir, Baloxavir marboxil and
Favipiravir (T-705) were purchased from MCE
15 3 6 2
H N O S : 386.0402; found: 386.0476. Oseltamivir
(https://www.medchemexpress.cn/). Tizoxanide was purchased from Key
Organics Ltd (Cornwall, UK).
CPE Inhibition Assay
The CPE inhibition assays with influenza viruses were performed in a 96-well
plate as described previously (28). Typically, MDCK cells were seeded and
grown for 24 h before infection. The growth medium was changed to virus
growth medium (DF-12) containing 2 μg/mL TPCK-trypsin. The tested
compounds were added to cells by a three-fold dilution (ranging from 0.005 μM
to 100μM) series in virus growth medium, and DMSO was added as control.
The cells were infected with influenza viruses at an MOI of 0.005 suspended in
DF-12 with 2 μg/mL TPCK-trypsin or were mock infected. After incubation at
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37℃ for 72 h, the antiviral effect of the tested compounds was measured
using a CellTiter-Glo cell viability assay (Promega), as described by the
manufacturer. The luminescence was read by a SpectraMax M5 microplate
reader (Molecular Devices). The 50% CPE protection concentration (EC50
was calculated by Origin 8 software (OriginLab Corporation, USA).
Cytotoxicity Assay
)
Compound toxicity was evaluated against MDCK using CellTiter-Glo cell
viability assay following the manufacturer’s instructions. The tested
compounds were added to cells by a two-fold dilution (from 0.78 μM to 200 μM)
series in virus growth medium, and DMSO was added as control. The
luminescence for each well was read by a SpectraMax M5 microplate reader.
The 50% cytotoxicity concentration (CC50) was calculated by Origin 8 software.
Plaque, HA titration and TCID50 Assay
Plaque assay: Confluent MDCK cells monolayers in 12-well plates were
incubated with 10-fold serial dilutions of virus for 1.5 h. The virus inoculum was
then removed, followed by overlaying with DF-12 medium containing 1%
agarose, 1 μg/mL TPCK-trypsin, 100 U/mL penicillin, 100 μg/mL streptomycin.
After 72 h incubation, cells were fixed with 4% formaldehyde for 4 h, then
stained with 1% crystal violet for plaques counting.
HA titration assay: The tested virus (100 μL) was diluted in PBS with a
two-fold dilution series in U-bottomed 96-well plate, and the volume of 50 μL
virus diluent from the last well were discarded. Equal volume of pre-prepared
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chicken erythrocytes (1% v/v in PBS) was mixed into each well with tested
virus. Subsequently, the mixture was incubated for 30 min at room temperature
to allow for hemagglutination to occur. The highest dilution with complete
agglutination was recorded, and the reciprocal of the dilution was determined
as HA titer.
TCID50 (50% tissue culture infectious doses) assay: Briefly, confluent
MDCK cell monolayers in a 96-well plate was washed with PBS, and then the
virus growth medium (DF-12) containing 2 μg/mL TPCK-trypsin was added to
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-8
each well. The tested virus was serially diluted by 10-fold from 10 to 10 , and
each dilution was inoculated into MDCK cells in 96-well plate. After 72 h
incubation, TCID50 was calculated using the Reed-Muench methods as
described previously (29).
IAV Replicon Assay
IAV replicon assay was assessed using a previously described method (28).
The inhibition activity of tested compounds was calculated by Origin 8
software.
NA Inhibition Assay
The NA-Star Influenza NA Inhibitor Resistance Detection Kit (Applied
Biosystems) was used to measure the inhibition of NA activity according to the
manufacturer’s instructions. NA from influenza PR/8 virus was used for this
assay. The IC50 of tested compounds and oseltamivir carboxylate were
calculated by Origin 8.0 software.
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Entry Assay of Pseudotyped Particle of IAV
HEK 293T cells were transfected with pNL4.3, pCAGGs H1N1-HA and NA (or
H5N1 or H7N9) at the ratio=10:1:1, using Lipofectamine 3000 for 12 h in 6-well
plates. The medium was replenished with DMEM containing 0.2% BSA, and
supernatants with pseudotyped particles were collected at 60
h
post-transfection. For entry assay, MDCK cells were seeded in a 96-well plate
for 18 h. The pseudotyped particles were incubated with tested drugs at a
concentration of 10 μM before inoculated for 2 h on MDCK cells. The firefly
luciferase activity was detected using a Bright-Glo Luciferase Assay System
(Promega).
HI Assay.
HI assay was performed as previously (28). Tested compounds were diluted in
PBS with a 3-fold dilution series from starting 100 µM concentration and then
mix 25 µL of series each compound dilution with an equal volume pre-prepared
4HAU PR/8 virus in U-bottomed 96-well plate. After 15 min incubation at room
temperature, 50 µL of freshly prepared chicken erythrocytes (1% v/v in PBS)
were added to each well with multichannel pipettes. Subsequently, the mixture
was incubated for 30 min at room temperature to allow for hemagglutination to
occur. PBS without virus and with virus alone were used as positive and
negative control, respectively.
Quantitative Real-Time PCR
Total cellular RNA was isolated from cell samples infected with IAV using the
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RNeasy Mini Kit (QIAEN). Absolute quantitation was performed using the ABI
Step One Plus platform with a One-Step PrimeScript RT-PCR Kit (Takara). The
details of probe sequences, primers were shown as follows: Probe with
sequence FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1; Inf-F with
sequence 5’-GACCRATCCTGTCACCTCTGAC-3’; Inf-R with sequence
5’-AGGGCATTYTGGACAAAKCGTCTA-3’. Relative Quantitative PCR (qPCR)
was performed using the ABI Step One Plus platform with a One-Step TB
Green® PrimeScript™ RT-PCR Kit (Takara) according to the manufacturer’s
instructions. Specific primers used for qRT-PCR were shown as follows:
GAPDH
(Fw,5’-CATGAGAAGTATGACAACAGCCT-3’;
Rev,5’-AGTCCTTCCACGATACCAAAG
(Fw,5’-AGACCATCGGCTGTTAATGG-3’;
T-3’),
HA
Rev,5’-TTGTGTATTGGGCG
TCTTGA-3’), NA (Fw,5’-TTCAGGCAGAATGAATGCAG-3’; Rev,5’-TGCGGA
AAGCCTAATTGAGT-3’), PB1 (Fw,5’- AGCGGGTATGCACAAACAGA-3’;
Rev,5’-ATAAGTCTGGCGACCTTGGG-3’). Data were normalized to the
-ΔΔCt
expression of human GAPDH. The 2
relative expression.
method was used to calculate
Time of Addition Assay
Time of addition assay was performed as previously described method (30).
Briefly, MDCK cells were seeded in a 6-well plate and incubated overnight and
then washed with PBS. Tested compounds at a concentration of 10 μM or
DMSO were added at following different time intervals before or after
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inoculation with PR/8 reporter virus (at 0 h): phase I, -2 to 0 h; phase II, 0 to 2 h;
phase III, -2 to 2 h; phase IV, 2 to 12 h; phase V, 0 to 12 h. At 12 h post
infection, the supernatants and cells were harvested. Virus titers were
assessed by plaque assay and qPCR.
Combinational Effects of Compounds 4a/4d and NAIs in vitro
MDCK cells in 96 well plates were infected with influnza virus PR/8 strain at
100 TCID50/well, followed by the addition of compounds 4a/4d and NAIs in
serial dilutions (for compounds 4a/4d, 0.16-10 μM; for zanamivir and
oseltamivir carboxylate, 1.23-100 μM;). After incubation at 37℃ for 3 days, the
antiviral effect was measured using a CellTiter-Glo cell viability assay
(Promega). Data were analyzed using MacSynergy II software.
Western Blot Assay
A549 cells seeded in 6-well plates were washed twice with PBS and infected
with PR8 virus at the MOI of 0.5. DF-12 medium containing 10 μM tested
compounds or DMSO was added to the cells at the same time. After incubation
for 24 h, the cells were lysed and loaded onto SDS-PAGE gel to eletrophoresis.
The proteins were then transferred to a polyvinylidene difluoride membrane.
After being blocked for 1 h, the membranes were washed and incubated with
anti-IAV HA, M1, PB1 (Invitrogen), NA (R&D systems) antibodies or
anti-GAPDH (Invitrogen) antibody as control at 4 ℃ overnight. The
membranes were washed and incubated with appropriate secondary antibody
at room temperature for 1 h. The proteins were detected using an
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electrochemiluminescence detection system.
Fluorescence Assay of Reporter Virus
A549 cells seeded in Black 96-well plates were washed twice with PBS and
infectied with PR8 reporter virus at MOI of 0.1. DF-12 medium containing
tested compounds or DMSO control was added to the cells at the same time.
At 2 h post-infection, cells were washed with PBS and medium containing
tested compounds were added. After 18 h incubation, cell images were
obtained via Zeiss Axio Observer inverted microscope. The following filters
were used: DAPI chanel for nuclear staining, GFP chanel for virus reporter.
Transcriptome Sequencing
Total cellular RNA was isolated from cell samples infected with IAV using the
RNeasy Mini Kit (QIAEN). The quality and quantity of RNA was analyzed by
Agilent 2100 Bioanalyzer (Agilent Technologies) and Nanodrop 2000 (Thermo
Scientific), respectively. RNA from each sample (5 µg) was used to create the
transcriptome libraries with IlluminaTruSeq™ RNA Sample Preparation Kit
(Illumina) following the manufacturer’s guidelines. And sequencing was
performed on IlluminaHiSeq™2500 platform. Q20 was used as a quality
control standard to filter the raw reads. Low quality reads were filtered while
the adaptors of high quality reads were removed, and the clean reads were
aligned with the reference genome using HISAT. No more than two
mismatches were allowed in the alignment.
Differential Expression and GO Enrichment Analysis
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Read count of genes was determined using Ht-seq method. Differentially
expressed genes (DEGs) were identified by DESeq2 with significant level at
adjusted P value = 0.05 which corrected applying Benjamini and Hochberg
False Discovery Rate (FDR) method (31). GO association data were
downloaded
from
the
Gene
Ontology
(GO)
database
(http://www.geneontology.org/) and enrichment analysis of biological
processes category was conducted by topGO (32). FDR correction was used
to identify significantly enriched GO terms and was also used when the FDR
was less than 0.05. All statistical analyses were performed using R 3.4.0
(www.bioconductor.org).
Lung Organoids Induction
hESCs maintenance: human embryonic stem cells (hESCs H9, provided by
Harvard university) were maintained on plate coated with mouse embryonic
fibroblasts (MEF) and cultured in hESC maintenance medium at 37℃ and 5%
2
CO .
Endoderm and anterior foregut endoderm induction: the induction was
performed as previously described (33, 34). Briefly, MEF were depleted by
passaging onto Matrigel supplied with maintenance media and incubated at
37℃ for 24 h. After MEF depletion, primitive streak and embryoid body
induction was perfomed in primitive streak/embryoid bodies formation media in
low attachment 24-well plates for 12-16 h. The media was replaced with
endoderm incuction media and incubated for 36-40 h. Embryoid bodies were
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kept supply with fresh medium every day and maintained in a humidified 5%
/5% CO atmosphere at 37°C. Endoderm yield was determined by the
O
2
2
expression of CXCR4. When the endoderm yield was more than 90%, cells
could be used for further induction. Embryoid bodies were digested with 0.05%
EDTA-trypsin and plated on fibronectin-coated multiple well plates on day 4.
Cells were incubated in Anteriorization media-1 for 24 h followed by switching
to Anteriorization media-2 for another 24 h.
Formation of lung bud organoids and branching morphogenesis: the
process was performed as previously described (33). At the end of anterior
foregut endoderm induction (after 24 h), cells were treated with ventralization
media for 48 h and three-dimensional clump formation was observed. The
clumps, called lung bud organoids (LBOs), were suspended by gently pipetting
and removed to ultra-low attachment 24-well plate with branching media. The
LBOs were fed every other day until d20–d25. The d20-d25 LBOs with
wrinkled structure were embedded in 100% Matrigel in 24-well transwell
inserts and incubated in incubator until the Matrigel solidified. Branching media
were added to the well, after which the transwell was inserted, branching
media added into the transwell insert as well. Media were changed every other
day.
Statistical Analysis
Data are presented as means ± standard deviations. Statistical significance
between 2 groups was estimated using the unpaired Student’s test or ANOVA
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for comparison of 3 groups and performed using GraphPad Prism 7 software.
P value of <0.05 were considered as difference significant.
Data Availability
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Results
Anti-influenza Activity of Novel Thiazolides.
To improve the anti-influenza virus activity of tizoxanide, we designed and
synthesized a total of 15 tizoxanide derivatives according to two strategies in
this study. The in vitro anti-influenza A virus (IAV) activity of all the compounds
1a-4d were evaluated in cytopathic effect (CPE)-based cell culture model that
utilizes a H1N1pdm09 strain (CA/07). The EC50 values were summarized in
Figure 1.
Based on Structure-Activity Relationship (SAR) analysis (Figure 1), we found
that the activities of derivatives decreased when the flexible side chain
substituents and aliphatic ring substituents were introduced into the N atom in
the amide (strategy I). The anti-IAV activity data indicated that the larger the
substituent group on the amide N atom, the lower activity. Introduction of
substituents on the amide N atom may not be conducive to the improvement of
activity. The antiviral activities of nitazoxanide and tizoxanide are comparable,
suggesting that the introduction of substituents into hydroxyl groups may be
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the direction of structure optimization. To verify this hypothesis, we introduced
flexible side chain substituents into hydroxyl groups (strategy II), and the
antiviral activity increased with the extension of flexible side chains. Four
derivatives showed 10-fold improved activities against H1N1pdm09 compared
with tizoxanide/nitazoxanide. The EC50 value of 4d was 0.16 μM with best
antiviral activity and 4a was also very active with the EC50 value of 0.62 μM.
Furthermore, the CC50 value of compounds 4a/4d against MDCK cells were
87.57±5.57 μM and above 200 μM, the selection index (SI) were 141 and
above 1250 for 4a and 4d, respectively. The optimization position and principle
was therefore outlined, and the preferred candidates 4a and 4d showed
significantly enhanced anti-influenza virus potentials as compared to
tizoxanide. The synthetic routes of compounds 4a/4d were shown in
supplementary scheme 1.
Compounds 4a and 4d Have Activity against Multiple Influenza Viruses In
Vitro.
To explore whether compounds 4a and 4d exert broad-spectrum anti-influenza
activities, the CPE protection assay was employed to evaluate the inhibitive
effects of the above compounds against representative IAV strains including
H1N1, oseltamivir-resistant H1N1, H3N2, H7N9 and influenza B viruses strains.
Results represented in Table 1 indicated that all strains were susceptible to 4a
and 4d, suggesting that the anti-influenza efficacy is not subtype specific. The
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anti-influenza PR/8, CA/07 and B type activities of compounds 4a/4d were
comparable to that of oseltamivir carboxylate. Moreover, compound 4a/4d
showed high antiviral efficacy against both naturally occurred NAI-resistant
strains and lab-generated NAI-resistant strain, indicating that 4a and 4d are
potential candidates for fight of NAI resistance.
The virus yield reduction assay was performed to further evaluate the inhibition
of compounds 4a/4d on IAV infection. Briefly, cells were incubated with
different concentration of compounds (2.5, 1.25, 0.625 μM) and DMSO control
at 37℃, and then four representative IAV strains (PR/8, JN/15 ZX/1109 and
HK/68) were inoculated into cells, respectively. At 24 h post infection, the virus
yields were determined by quantitative real-time PCR (qRT-PCR) for
intracellular viral RNA and plaque assay for supernatant viral particles. As
shown in Figure 2A and 2B, compounds 4a/4d reduced the intracellular viral
RNA in a dose-dependent manner. Both compounds significantly decreased
(P<0.05) the infectious virus particle propagation in cell supernatants in a
dose-dependent manner for all tested strains, shown in Figure 2C and 2D.
We next examined the effect of compounds 4a and 4d on viral protein
generation during the infections by fluorescence assay and western blot assay.
We generated a virus strain with GFP reporter PR/8-NS1-GFP, NS1 gene
tagged with a GFP gene, to track the dynamic activity of virus. Briefly, MDCK
cells were infected with PR/8-NS1-GFP reporter virus at a multiplicity of
infection (MOI) of 0.1 in the presence of compounds 4a and 4d at indicated
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concentrations. The fluorescence imaging showed that generation of viral NS1
protein was strongly inhibited in presence of compounds 4a and 4d (Figure 3A).
The western blot experiment was carried out on A549 cells. The results
revealed that the expression of viral HA, NA, M1 and PB1 proteins were
significantly attenuated in presence of compounds 4a and 4d (Figure 3B and
3C). Consistently, the mRNA level of viral HA, NA, M1 and PB1 were reduced
(Figure 3D). Herein, the inhibition of viral protein generation was in accordance
with reduction of viral mRNA level.
Compounds 4a/4d and NAI Synergistically Inhibit Oseltamivir-resistant
Influenza Virus Strain in vitro
The synergetic effects of compounds 4a/4d with NA inhibitors (oseltamivir
carboxylate and zanamivir) were investigated using an 8*6 combinatorial
design (35). Combination of drugs led to enhanced inhibition of virus-induced
CPE in MDCK cells, as reflected by the volumes of the area above the
expected inhibitory effect for each drug independently (Figure 4) by
MacSynergy II analysis (35). The resulting surface of merely additive
interaction would appear as a horizontal plane at 0% inhibition from expected.
2
The values of synergy/antagonism volumes under 25 μM % were regarded as
2
2
insignificant at 95% confidence; values between 25 μM %-50 μM % should be
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considered as minor significant; values between 50 μM %-100 μM % were
2
regarded as moderate significant and over 100 μM % indicated strong
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significant interaction. The net volumes of synergy resulting from combined
treatment of compound 4a and oseltamivir carboxylate or zanamivir were
122.2 and 372.9, respectively, indicating a strong synergistic effect of 4a and
NAIs (Figure 4A/4B). The net volumes of synergy resulting from combined
treatment of compound 4d and oseltamivir carboxylate or zanamivir were 76.1
and 128.7, respectively, indicating a moderate/strong synergistic effect of 4d
(Figure 4C/4D). These results suggested that compounds 4a/4d might be
potential options in fighting against drug resistance of influenza.
Compounds 4a/4d Inhibit the Intracellular Events of Influenza Virus
Infection.
Previous research showed that nitazoxanide and its active metabolite
tizoxanide inhibit the replication of IAV strain by selectively blocking the
maturation of the viral HA and impairing HA intracellular trafficking at
post-translational level (21). Whether compounds 4a/4d with improved
anti-influenza activity share the same molecular mechanisms needs to be
elucidated.
To investigate which stage of the influenza infection cycle was affected by
compounds 4a/4d, we performed time of addition experiment using PR/8
reporter virus (Figure 5A). Compounds 4a/4d inhibited influenza virus yields
when added concurrently with IAV infection or after infection (Figure 5B and
Figure S1) as well as control drug favipiravir that acts at viral genome
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replication stage, suggesting that 4a/4d acted at late phase of viral infection
cycle.
To further verify whether the compounds had effect on entry, we carried out
influenza pseudovirus assay (36, 37) with three kinds of pseudotyped particles
(H1N1, H5N1 and H7N9). The blockage of pseudotyped particles entry would
reduce the luciferase activities. An IAV specific HA neutralizing antibody (NAb)
or DMSO were used as positive or negative control, respectively. The results
revealed that compounds 4a/4d had no effect on the entry of pseudotyped
particles (Figure 5C). This finding was further verified by the results obtained
from a hemagglutination inhibition (HI) assay (38) (Figure 5D). No inhibition of
hemagglutination were observed under test concentration, indicating that the
compounds 4a/4d were unable to inhibit the absorption of IAV into host cells.
To address the possibility that compound 4a/4d inhibited the post-entry events,
influenza replicon assay was performed alongside the positive control drug,
favipiravir, which inhibits the RNA-dependent RNA polymerase (RdRp) of
influenza virus (Figure 5E). As expected, compounds 4a/4d inhibited replicon
with EC50 value of 3.68 ± 2.19 μM and 18.87 ± 7.37 μM, respectively. We then
asked if the compounds prevent the NA-dependent progeny virus releasing,
and the NA inhibition assay was performed. As shown in Figure 5F, neither
compound 4a nor 4d was able to disturb cleavage of the substrate by NA from
influenza PR/8 in the test range. In contrast, oseltamivir carboxylate, as the
positive control, inhibited viral NA activity with an IC50 value of 2.57 ± 0.75 nM.
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Herein, we concluded that the compounds 4a/4d acted at the stage between
post-entry and viral particle release, rather than virus entry and release.
Transcriptome Analysis of 4a/4d.
Compounds 4a/4d inhibited the post entry event of virus and then the
molecular antiviral mechanism was explored via transcriptome analysis. A549
cells were infected with PR/8 virus for 8 h at MOI of 1 while compounds 4a/4d
(10 μM) or DMSO were added to the cells simultaneously. We isolated total
RNA in three independent experiments from the compound treated cells and
DMSO treated cells and built up sequencing libraries to perform deep
sequencing. Total mRNA profiles were analyzed. The expression of genes
2 2
with > 1.5Log FC (upregulation) or < -1.5Log FC (downregulation) were
defined as differentially expressed. As shown in Figure 6A and 6B, a total of
618 and 446 differentially expressed genes (DEGs) were identified from
compound 4a and 4d treated cells, respectively. We noticed that 250 DEGs
were overlapped between samples treated with compound 4a and 4d (Figure
6C). By importing datasets representing genes with differential expression
obtained from RNA-seq analysis for GO enrichment, we examined possible
biological function of DEGs and determined component complex (CC) or
biological process (BP) involved in antiviral mechanism of compounds 4a/4d in
cells. The results of GO analysis of the ten most significant subclasses of BP,
CC and molecular function (MF) were presented in Figure 6D according to
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enrichment ratio. It was revealed that the most enriched CC mainly include the
transcription AP-1 complex, transcription factor complex, and CHOP-ATF3
complex, and the most significant MF mainly include the MAP kinase
tyrosine/serine/threonine phosphatase activity, nuclear receptor activity, and
transcription factor binding. The subclasses of above DEGs were shown in the
heat map (Figure 6E). The known genes with antiviral potential, such as PTX3
and SERPINE1, were identified in the map. This might provide insights into the
antiviral mechanism of 4a/4d, and shed light on clues for search of novel
anti-influenza drug targets.
Compounds 4a/4d Inhibit Influenza Virus Infection in Human Lung
Organoids.
Human organoids system provided a novel platform to carry out in vitro drug
evaluations in a simulated in vivo condition. Human lung organoids have
certain substructures as human lungs, featured as alveolar epithelial cells,
goblet cells and basal cells. Human lung organoid could better simulate the
cellular heterogeneity of human organs. Increasing evidence suggested that
organoids are superior models for research of important pathogens, such as
influenza, Zika virus, Enterovirus, et al (39-41). To further explore the
therapeutic potential of 4a and 4d, we adopted lung organoids differentiated
from hESCs to mimic influenza infection in human. After 104 days of
development in three-dimensional culture, organoids were infected with
influenza PR/8 and HK/68 and simultaneously treated with compounds 4a/4d
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(10 μM), favipiravir (50 μM) or DMSO for 1h. At 24 h post infection, influenza
virus in lung organoids was quantified by qRT-PCR (Figure 7). The results
demonstrated that compounds 4a/4d had comparable antiviral efficacy with
positive drug favipiravir. Moreover, in terms of inhibition of seasonal influenza
H3N2, 4a/4d were more potent than favipiravir.
Discussion
The history of anti-influenza drug development witnessed the coevolution of
viruses and antiviral drugs. As the first generation of anti-influenza drug
including amantadine and rimantadine fade out history, researchers are more
and more concerned about the emergence and spread of drug resistance of
NAIs which is currently the first-line drug for influenza. Compared with
virus-targeted drugs, host-targeted drugs have lower probability of drug
resistance. NTZ was recently identified as a broad-spectrum antiviral drug and
has been repurposed for the treatment of influenza. NTZ was reported to target
host proteins and obstruct the maturation of HA of influenza virus. However,
the specific target of NTZ was not identified yet, and this drug has varied
antiviral mechanisms for different viruses, which made optimization of NTZ
difficult. We first tried to enlarge the space occupation of the N atom in the
amide, but the antiviral activity was not improved accordingly. On the contrary,
the antiviral activity decreased as the enlargement of the amide N position. We
therefore conclude that this position is not an ideal optimization target.
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Fortunately, a substantial increase of antiviral activity was observed while the
space occupation of hydroxyl group was improved. Based on this finding, we
move forward to extend this group, and the antiviral activity further increased
as the elongation of side chain. The optimization position and principle was
therefore outlined for influenza, and the novel structured candidates 4a and 4d
was chosen for in-depth antiviral research.
We explored the antiviral effects of 4a/4d against a variety of influenza virus
strains, including oseltamivir-resistant strains. Moreover, we explored the
synergetic effects of 4a/4d, which highlighted their potentials as future
anti-influenza drug candidates. The synergetic effects of compounds 4a/4d
when administered in combination with zanamivir or oseltamivir carboxylate
were revealed using a cell model infected with natural oseltamivir-resistant
isolate ZX1109. These findings provided potential strategies to combat
NAI-resistant strains in the future.
Any structural modifications of a compound may change its mode of action or
target. Therefore, we explored how compounds 4a/4d exert antiviral activities.
Based on a series of experiments, we narrowed the action stage of 4a/4d to an
intracellular period between post entry and viral release. We employed
transcriptome analysis and found a series of cellular events might contribute to
the antiviral activity of 4a/4d. Notably, the subset transcription factor AP-1
complex (P=0.00046), including DDIT3, FOS/FOSB, was significantly
over-represented. It was reported that influenza viral NS1 protein antagonized
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virus-induced activation of stress response by downregulating AP-1-dependent
gene expression (42). Here, we found the treatment of compounds 4a/4d
increase the expression of AP-1 complex, which may contribute to antiviral
activity. Interestingly, it was revealed that treatment of compounds 4a/4d
inhibited virus infection by modulation of a term of cytoplasm (P=0.001) related
genes, indicating that these cytoplasm related genes widely participated
post-entry steps of influenza virus infection such as protein translation,
assemble and transport. Significant upregulation of reported anti-influenza
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genes including pentraxin 3 (PTX3, P=3.29×10 ), SERPINE1 (P=1.89×10 ),
-
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ANKRD1 (P=4.19×10 ), PER1 (P=2.84×10 ) and NR4A3 (P=1.66×10 )
were detected in our analysis. PTX3 was reported to mediate antiviral activity
by modulating host immune in vitro and in vivo. By recognizing of HA
glycoprotein, PTX3 could play a role in virus neutralization and anti-influenza
activity (43). SERPINE1/PAI-1 was reported to inhibit IAV spread by inhibiting
glycoprotein cleavage (44). ANKRD1 was involved in IRF3-mediated antiviral
innate immune signaling pathway by enhancing induction of type I and type III
interferons (45). NR4A3 is a member of nuclear receptor subfamily. It is
reported that nuclear receptor may be involved in induction of amino acid and
lipid metabolism, potentially required for recovery from influenza infection (46).
Per-1, encoding by PER1, is a novel negative regulator of HIV-1 transcription
(47). It is suggested that up-regulate expression of PER1 regulated influenza
virus infection through the same mechanism. The above findings suggested
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that compounds 4a/4d might play a role in anti-influenza virus by regulating
intracellular transcription, immune system and/or lipid metabolism. These
genes or biological processes may provide novel target for future anti-influenza
research.
For the host-targeted drugs such as NTZ, the evaluation model was critical for
a comprehensive judgment of efficacy. The lung organoids we employed in this
research were derived from hESCs, which could better simulate the infection
of influenza virus to human (33, 48). The effectiveness of 4a/4d in lung
organoids further strengthened our confidence that the two candidates may
work in human tissue.
To conclude, this research discovered the optimization principle of NTZ as an
anti-influenza drug, and reported a series of thiazolides with relatively
broad-spectrum antiviral potential.
Acknowledgments
This work was supported by NSFC (81773631, 81703414), the National
Science and Technology Major Projects “Major New Drugs Innovation and
Development” (2018ZX09711003), and China Postdoctoral Science
Foundation (2018M633678).
Author Contributions
R.C. and W.Z. conceived and directed the research. L.Z., Y.Y, Q.D. and X.L.
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designed and performed most of the experiments. K.X. and G.Z. performed the
IAV replicon assay. K.Y. and Q.X performed the organoids-based experiments.
W.L., X.G., J.Y., and Y.L. helped to perform experiments or contribute to
reagents. W.Z., R.C. and L.Z. analyzed the data and wrote the manuscript.
Declaration of Interest Statement
No conflict of interest was reported by the authors.
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