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A small-molecule compound has anti-influenza A virus activity by acting as a ‘‘PB2 inhibitor” Teng Liu, Miaomiao Liu, Feimin Chen, Fangzhao Chen, Yuanxin Tian, Qi Huang, Shu-Wen Liu, and Jie Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00531 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Molecular Pharmaceutics

A small-molecule compound has anti-influenza A virus activity by acting as a ‘‘PB2 inhibitor” Teng Liu, Miaomiao Liu, Feimin Chen, Fangzhao Chen, Yuanxin Tian, Qi Huang, Shuwen Liu*, Jie Yang* Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China KEYWORDS. Influenza A virus; Virus replication; Viral polymerase; PB2; PB2 cap-binding inhibitor.

ABSTRACT. With regular influenza epidemics and the prevalence of drug-resistant influenza virus strains, it is extremely crucial to develop effective and low-toxicity anti-influenza A virus drugs that act on conserved sites of novel targets. Here, we found a new anti-influenza virus compound, 1,3-dihydroxy-6-benzo [c] chromene (D715-2441), from a library of 8,026 small molecule compounds by cell-based MTT assay and explored the underlying mechanisms. Our results revealed that D715-2441 possessed antiviral activities against multiple subtypes of influenza A viruses (IAVs) strains, including H1N1, H5N1, H7N9, H3N2, the clinical isolate 690 (H3) and oseltamivir-resistant strains with the H274Y NA mutation, and suppressed the early steps in the virus replication cycle. Further mechanistic studies indicated that D715-2441 clearly inhibited viral polymerase activity and directly influenced the location of the PB2 protein. Moreover, binding affinity analyses confirmed that D715-2441 bound specifically to the PB2cap

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protein. Further, protein sequence alignment and a computer-aided molecular docking indicated that highly conserved amino acid residues in the cap-binding pocket of PB2cap were possible binding site of D715-2441, which indicates that D715-2441 might be employed as a cap-binding competitor. Moreover, the combination of D715-2441 and zanamivir possessed a remarkable synergistic antiviral effect, with a FICI value of 0.40. In conclusion, these results strongly suggest that D715-2441 has potential as a promising candidate against IAV infection. More importantly, our work offers novel options for the strategic development of PB2cap inhibitors of IAV.

INTRODUCTION It has resulted in high morbidity and mortality in humans and several animals about the ongoing influenza A virus seasonal epidemics, pandemics and worldwide outbreaks, which pose a serious threat to the economy as well as public health

1-4

. For instance, approximately 50

million people caused the death of the “Spanish” influenza virus (H1N1) pandemic of 1918 5. The extensive spread of the pandemic influenza A H1N1 2009 virus (A/2009/H1N1) in 214 countries have resulted in the deaths of at least 18,000 people 6. Recently, novel reassortant avian influenza strains, such as H5N8, H10N8 and H7N9, have generated and circulated in Asia 7. In particular, some studies have indicated that avian influenza viruses H7N9 and H5N1 strains might break in the species barrier and then lead to infection in humans

8-9

, thus posing a great

threat to public health later. Hence, flu prophylaxis and early treatment have gained much importance. Generally, vaccination is one strategy for controlling influenza infections. However, live attenuated and inactivated influenza vaccines often show a decrease or even loss of productivity


2

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and efficiency because of antigenic drift and shift. Therefore, development of more efficient and timely vaccines to overmaster flu outbreaks remains a significant challenge

10-11

. Another

treatment strategy is the application of anti-influenza virus drugs. According to different targets, there are the five classes of anti-influenza agents, including NA inhibitors, M2 ion channel blockers, RNA polymerase inhibitors, inosine 5’monophosphate dehydrogenase inhibitors and interferon and small interfering siRNAs 12. Unfortunately, the increasing number of reports have been published in recent years concerning drug-resistant variants of influenza viruses, especially oseltamivir-resistant strains of IAVs. For example, mutations of oseltamivir at amino acid residues E119, R292 and N294 in N2 and H274 in N1 have resulted in resistance

13-15

, limiting

the development and effectiveness of this drug. Furthermore, patients receiving M2 ion channel blockers or NA inhibitors for IAV infection may be at risk of severe side effects. For instance, prolonged use of amantadine has potential central nervous system toxicity

16

. Therefore, new

therapeutic drugs are urgently needed to combat influenza virus, especially those with novel modes of action. The influenza viral vRNP is released into the cytoplasm of target cells in the early stages of virus infection, followed by transport to the nucleus in the form of active transport, and then transcription and replication of the virus genome initiated. As far as we know, transcription of influenza virus occurs via a special mechanism

17

. Influenza RNA polymerase cannot directly

synthesize the cap structure of virus mRNAs, which is consistent with three different gene fragment, including polymerase acidic protein (PA), polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2). Instead, viral mRNAs transcription is initiated by influenza PB2 capturing the cap structure of the host's mRNA, which is known as the ‘cap-snatching’ mechanism and is dominated by the cap-binding domain (amino acid residues 318–483) of the


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PB2 subunit (PB2cap). In this course, PB2cap joins to the 5’-cap structure of a target cell premRNA molecule, followed by snatched pre-mRNA 10-13 nucleotides with PA endonuclease activity located downstream of cap cleavage

18-20

. Subsequently, viral PA, PB1, PB2 and NP

proteins are newly synthesized on the template of viral mRNA in the cytoplasm and need to enter the cell nucleus to assemble into the new vRNP with virus RNA

21

. Therefore, PB2cap plays a

critical role in viral replication stage, suggesting that it might catch the attention as a target for the expansion of antiviral drugs. More importantly, cap-binding domain of the PB2 subunit is reasonably conserved among different subtypes

22-23

, and thus, antiviral drugs that target the

functional domains are likely to exhibit cross-subtype anti-influenza efficacy. In an effort to screen small molecule inhibitors that specifically bind the above viral gene, we found a compound, namely, 1,3-dihydroxy-6-benzo [c] chromene (D715-2441), by highthroughput screening from a compound library composed of 8,026 compounds using a cell-based MTT assay. D715-2441 exhibited a significant inhibitory effect on IAVs in vitro. Kock et al.

24

reported the molecular structure of D715-2441, but its application in anti-influenza was first shown in the present work. Here, we explored the potential mechanisms of antiviral activity of D715-2441 against influenza viruses and indicated that D715-2441 may be a new lead compound for the growth of novel inhibitors of influenza PB2.

MATERIALS AND METHODS

Cells, viruses and agents Madin Darby canine kidney (MDCK) cells, human lung epithelial (A549) cells, and human embryonic kidney (293T) cells were purchased from the American Type Culture Collection


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(ATCC, Manassas, USA). MDCK and 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) assisted in 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). A549 cells were cultivated in RPMI1640 medium assisted in 1% penicillin/streptomycin and 10% FBS. After contracting the virus, the infected cells were maintained in FBS-free medium added in 1 µg/mL TPCK trypsin. Influenza A viruses strains, including A/Puerto Rico/8/34 (H1N1),

A/FM-1/47

(H1N1),

Vietnam/1194/2004

(H5N1),

A/Anhui/1/2013

(H7N9),

A/Aichi/2/68 (H3N2), oseltamivir-resistant influenza A (H1N1) viruses with NA-H274Y mutation and the influenza A virus 690 (H3), which is a clinically relevant virus, were applied in this work. Viral stocks were readied in 9-day-old embryonated eggs, and then allantoic fluid including influenza was stored at -80 °C in aliquots and quantified using plaque assay. D7152441 (229 g/mol) was purchased from TopScience Company with 98% pureness (Shanghai, China). Ribavirin and zanamivir were purchased from Sigma-Aldrich (St. Louis, MO, USA). PB2-19 and CL-385319 were synthetized with a pureness of more than 98% in our laboratory.

Plasmids HIV-1 backbone plasmid encoding Env-defective and luciferase-expressing (pNL4-3.luc.R-E),

VSV

glycoprotein-encoded

plasmid,

A/Thailand/Kan353/2004-HA

and

A/Thailand/Kan353/2004-NA were generously provided by Professor Frank Kirchhoff (University Ulm, BW, Germany). The pHW2K-PB1, pHW2K-PB2, pHW2K-PA, pHW2K-NP and a firefly luciferase reporter plasmid (pPolI-Fluc) were kindly provided by Professor Bojian Zheng (University of Hongkong, Hongkong). A Renilla luciferase plasmid (hRluc-TK) was obtained from Promega (Beijing, China).


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Cytotoxicity assay The cytotoxicity of D715-2441 was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT; Sigma, USA) assay

25

. Briefly, 2×104 MDCK cells each well were

seeded into a 96-well tissue culture plate and cultivated overnight until they had grown to 90% confluence. Serially diluted D715-2441 or blank control solutions (0.1% DMSO) were supplemented to cells. After 48 h of incubation, MTT solutions were added and cultivated for 4 h at 37 °C. Prior to detection, in order to dissolve the formed formazan crystals we employed 150 µL DMSO added to well. The absorbance was recorded using microplate reader (GENios Pro, TECAN, Bedford, MA, USA) at 570 nm. The concentration of D715-2441 resulted in the death of 50% cells was regarded as the 50% cytotoxic concentration (CC50) and calculated using the CalcuSyn software.

Antiviral assay and microscopy In order to evaluate inhibitory activity of D715-2441 against influenza A virus, we utilized viruses to infect MDCK cells using a 0.01 multiplicity of infection (MOI) for 1 h, including A/Puerto

Rico/8/34

(H1N1),

A/FM-1/47

(H1N1),

A/Vietnam/1194/2004

(H5N1),

A/Anhui/1/2013 (H7N9), A/Aichi/2/68 (H3N2), oseltamivir-resistant influenza A (H1N1) viruses with NA-H274Y mutation and the clinical isolate 690 (H3). Subsequently, cells were washed twice with PBS to eliminate unabsorbed influenza. D715-2441 was then added into the infected cells and cultured for 48 h. The antiviral effect of D715-2441 was quantitatively evaluated with cell-based MTT assay as previously described. Additionally, the cytopathic effect (CPE) induced the virus was observed by microscopy. The experiment was performed three times and ribavirin was applied as a positive control.


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Plaque assay Confluent monolayers of MDCK cells were adsorbed A/Puerto Rico/8/34 (H1N1) virus for 1 h at a 0.01 MOI. After getting rid of unabsorbed virus, the virus-infected cells were incubated in 1.5 mL of 2×MEM including 1 µg/mL TPCK (Sigma-Aldrich, USA), 2% agar (Sigma-Aldrich, USA) and various concentrations of D715-2441 for 48~72 h as previously described 26. In order to stain with crystal violet (Sigma-Aldrich, USA), we applied 4% paraformaldehyde to fix cells. Subsequently, inhibition of D715-2441 on influenza viral plaque formation was observed by the number of plaques.

Western blot analysis MDCK cells were adsorbed influenza virus and then added with D715-2441 at serial concentrations as described above. At 24 h post-infection (pi), cells were lysed and gathered. The protein samples were separated using 10% SDS-PAGE. Hereafter, the protein bands were transformed into polyvinylidene difluoride (PVDF) membrane and cultured overnight with primary antibodies against influenza NP (1:2000 dilution, Abcam, Cambridge, MA, USA) and βactin (1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA) as control at 4 °C. And it was cultivated using anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Finally, chemiluminescent signal was detected with Lumiglo reagent (Cell Signaling Technology, Beverly, MA, USA) and a FluorChem E imaging system (ProteinSimple, Silicon Valley, CA, USA). Three independent experiments were repeated.


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Quantitative real-time PCR assay The expression of different gene was measured by quantitative real-time PCR (RT-PCR) as previously reported

27-28

. In brief, a confluent cell monolayer was treated with D715-2441 at the

indicated concentrations for 24 h after infecting with A/Puerto Rico/8/34 (H1N1) virus. Total RNA in cells was abstracted by TRIzol (Invitrogen, Carlsbad, CA, USA) and then reversetranscribed into cDNA utilizing the PrimeScript RT reagent kit (TaKaRa, Dalian, China). RTPCR was implemented in an ABI7500 system (Applied Biosystems, Foster, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as the reference gene. The relative expression of the genome was calculated using the classical 2-∆∆CT method with 7500 software. In addition, the expression of influenza HA was detected to observe the duration of antiviral activity of D715-2441 at 24, 48, and 72 h pi. Ribavirin was applied as a control. A similar experiment was performed utilizing the A549 cell to confirm there was no cell line selectivity for the antiviral effect of D715-2441. All experiments were performed at least three times.

The

HA

gene

of

the

primer

sequences

was

as

follows:

5’-

TTCCCAAGATCCATCCGGCAA-3’ (forward) and 5’-CCTGCTCGAAGACAGCCACAACG3’

(reverse),

and

the

sequences

for

GAPDH

gene

were

as

follows:

5’-

AGGGCAATGCCAGCCCCAGCG-3’ (forward) and 5’-AGGCGTCGGAGGGCCCCCTC-3’ (reverse).

Immunofluorescence microscopy Immunofluorescence microscopy was conducted with simple alterations as previously reported 29

. In Brief, we utilized PBS to wash MDCK or A549 cells twice after 24 h pi, and then cells

were fixed with 4% paraformaldehyde for 20 min and blocked with 3% albumin from bovine


8

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serum for 1 h. The cells were incubated overnight with antibody against influenza NP protein (1:250 dilution, Santa Cruz, CA, USA) or antibody against influenza PB2 (1:500 dilution, GeneTex, USA) at 4 °C, followed by incubation a fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:250 dilution, Santa Cruz, CA, USA) or a Alexa Fluor®488-conjugated anti-rabbit IgG antibody (1:500 dilution, Cell Signaling Technology, Beverly, MA, USA) for another 1 h. The samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and next captured on a confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss Jena, Germany) and a Ti-Eclipse inverted fluorescence microscope (Nikon, Tokyo, Japan) for 24 h pi.

Time-of-addition assay To probe which stage was affected by D715-2441, we chose two types of time-of-addition studies as previously reported with minor modifications 30. Various concentrations of D715-2441 were pre-cultivated with A/Puerto Rico/8/34 (H1N1) virus for 30 min. The mixtures were transferred to MDCK cells for another 1 h, which was termed pre-infection. After removing the mixtures, cells were incubated in FBS-free medium. Post-infection, the virus-infected cells were added with D715-2441 as described for the “antiviral assay” section. Entire infection meant cells were handled as described for the “pre-infection”. Subsequently, the culture supernatants were replaced with FBS-free medium including D715-2441 for 48 h. The inhibition activity of D7152441 against influenza in three modes of additional assays was detected by the MTT assay as depicted above. By contrast, we profoundly researched the stages of the influenza life-cycle that was impacted by D715-2441. Briefly, infected MDCK cells were incubated with 20 µM D7152441 at the different time intervals (0−2, 2−5, 5−8, 8−10 and 0−10 h), which covered the whole


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replication cycle of influenza virus. Influenza A virus life cycle was roughly divided into four time intervals from approximately 10 h after virus infection, including early replication of virus genome (0-2 h, 2-5 h), late replication of the genome (5-8 h) and progeny virus particle release (8-10 h). After incubation for 10 h pi, the expression of influenza NP protein and HA mRNA were measured by western blotting or RT-PCR as mentioned above.

Assessment of synergistic antiviral effects of compounds The possible synergistic antiviral activity of D715-2441 combined with zanamivir was conducted as described previously

31

. The virus-infected MDCK cells were cultured with the

indicated concentrations of D715-2441 and zanamivir in combinations or alone. After 48 h, the IC50 of each combination against influenza virus was measured using cell-based MTT assay as depicted above. Afterwards, the fractional inhibitory concentration index (FICI) was computed using the following formula: FICI = [(IC50 of the compound in combination)/(IC50 of the compound alone)] + [(IC50 of zanamivir in combination)/(IC50 of zanamivir alone)]. FICI < 0.5 was regarded as an obviously synergistic antiviral effect.

Pseudovirus neutralization assay We employed NA and HA plasmid of A/Thailand/Kan353/2004 (H5N1) and capsid protein from HIV to construct influenza H5N1 pseudovirus as previously reported

27

. Briefly, 1 µg of

each of A/Thailand/Kan353/2004-HA and A/Thailand/Kan353/2004-NA and 3 µg of HIV backbone plasmid (pNL4-3.luc.R−E−) were co-transfected into 293T cells by polyetherimide (PEI). The supernatants were harvested after 48 h for single-cycle infection. VSVG pseudovirus as a control was generated in the similar step as H5N1 pseudovirus except a VSV glycoprotein-


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encoded plasmid was used to replace the influenza NA and HA. The pseudovirus titers were tested by the luciferase assay (Promega, Madison, USA). The pseudovirus H5N1 or VSVG was cultivated with D715-2441 at serial concentrations for 30 min. Hereafter, the mixtures were put in MDCK cells for 48 h. Infected-MDCK cells were lysed for 20 min and the lysis solution was converted to 96-well white polystyrene microplates. The relative luciferase values was detected by the Ultra 384 luminometer (GENiosPro, TECAN, Bedford, MA, USA) after the addition of luciferase substrate. CL-385319 was utilized as a control as previously reported 32.

Neuraminidase (NA) inhibition assay NA enzyme inhibition test was implemented to estimate the effect of D715-2441 on the release of influenza progeny virion as described previously

33

. Briefly, 15 µL of A/Puerto Rico/8/34

(H1N1) virus solution was mixed with 5 µL of serially diluted D715-2441 in a 96-well black plate for 30 min. Zanamivir was used as positive control. Next, 30 µL of MU-NANA (2-(4methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium; Sigma-Aldrich, USA) matrix solution dissolved in diluted buffer (4 mM CaCl2 and 32.5 mM MES, pH 6.5) was put in each well and cultivated for 1 h in the dark. Subsequently, 50 µL of 14 mM NaOH was put in the above reaction solution to terminate the reaction. We utilized an excitation wavelength of 340 nm and emission wavelength of 440 nm to measure the fluorescence intensity of the outcome 4methylumbelliferone using a microplate reader. The inhibition rate of NA enzyme activity was computed by the following formula: inhibition rate (%) = (Fvirus−Fsample)/(Fvirus−Fsubstrate) × 100%.


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Minireplicon assay The influence of D715-2441 on the activity of influenza viral ribonucleoprotein complex (vRNPs) was detected by a minireplicon assay as depicted by Yuan et al 34. In brief, 293T cells (60~70% confluent) were co-transfected with 50 ng of one of pHW2K-PB1, pHW2K-PB2, pHW2K-PA, pHW2K-NP and firefly luciferase reporter plasmid (pPolI-Fluc) and 10 ng Renilla luciferase plasmid (hRluc-TK) (Promega, Madison, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as a transfection reagent. After 5 h of transfection, the supernatant was substituted with DMEM with 0.5% FBS and serial diluted D715-2441, zanamivir or 0.1% DMSO. Upon lysing the cells to obtain cell lysates (Promega, Madison, USA) for 20 minutes, polymerase activity was examined at 24 h pi.

Surface plasmon resonance (SPR) measurements The combining affinity between virus PB2cap protein and D715-2441 or PB2-19

35

was

analyzed using the PlexArray® HT system (Plexera® Bioscience, Beijing, China) under the conditions described in our previous paper with small changes

36

. After that influenza PB2cap

proteins were fixed on a sensor chip external by photo-cross-linking, various concentrations of D715-2441 were injected as analytes at a flow of 3 µL/s. The contact time and dissociation time were 300 s, respectively. The running buffer was 10 mM phosphate buffer saline with Tween-20 (137 mM NaCl, 2.7 mM KCl, 0.05% Tween-20, PH 4.5). We used glycine−HCl (pH 2.0) and the phosphate buffer to regenerate and wash, respectively. The KD value was figured with PlexeraDE software by curves fitted through the Langmuir equation.

Fluorescence polarization (FP) assay


12

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The FP test was performed in 96-well black micro-plates (Greiner Bio-One, Germany) using FITC-labelled m7GTP (EDA-m7GTP-ATTO 488; Jena Bioscience, Germany) as a detection probe

37

. Briefly, the mixtures of PB2cap (1 µM) and FITC-m7GTP (20 nM) dissolved in

reaction buffer were dispersed into each well for 30 min. Next, the increasing amounts of D7152441 (0–200 µM in 50 µL) were put in the above mixtures for 30 min. The fluorescent signals were then gauged using Infinite M1000 Pro (TECAN, Switzerland). The reaction and dilution buffer comprised 50 mM HEPES (pH 7.2), 100 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT) and 1% DMSO.

Molecular docking The structure of PB2cap was downloaded from the RCSB protein data bank (4CB4). Prior to molecular docking, PB2cap protein and D715-2441 were assigned AMBER ff14SB force field and AM1-BCC charges, respectively. The three-dimensional structure of the PB2cap-D715-2441 complex was derived using UCSF Dock 6.7. The electrostatic interactions and van der Waals interactions between D715-2441 and binding amino acid of PB2cap were calculated and constituted the Grid scores. Thereafter, the optimal conformations were received by cluster analyses (RMSD threshold of 2 Å). The Protein-Ligand Interaction Profiler was utilized to analyze the simulated data. The conservation of D715-2441 binding amino acids in avian, swine, and human IAV was analyzed using a Python script.

Synthesis of D715-2441 D715-2441 was synthesized as depicted by Kock et al.24 with small modifications. Phloroglucinol (2.0 molar amount) and o-bromobenzoic acid (1.0 molar amount) were put in a


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round-bottomed flask and then sodium hydroxide solution (2.0 molar amount dissolved in water) was added dropwise followed by placement of a stir bar. The experimental device was plugged with a piston, and then a balloon was attached, followed by heating on a thermostatically heated magnetic stirrer with stirring until solutions were stirred at 60 °C for 15 minutes. Copper sulfate (10%, 0.5 mL) was put in dropwise to the flask during the thermal reaction, and the reaction was maintained under positive pressure of argon. Heating was carried at 95 °C, followed by stirring for 3 hours and then stirring at 37 °C overnight. The stirring was stopped the next day, and the temperature was reduced to room temperature. Finally, to obtain the crude product, the precipitate was filtered out with suction, washed with distilled water, and dried under a vacuum and high pressure.

1

H NMR (400 MHz, DMSO) δ 10.91 (s, 1H), 10.15 (d, J = 1.0 Hz, 1H), 8.97 (d, J = 8.4 Hz, 1H),

8.20 (d, J = 7.9 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 6.41 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H). 13

C NMR (101 MHz, DMSO) δ 161.15, 159.64, 158.10, 153.74, 135.81, 135.40, 129.77, 126.78,

126.21, 118.81, 100.18, 99.04, 95.48. HRMS (ESI) calcd for C13H9O4 [M+H]+: 229.0495. Found: 229.0492.

Statistical analysis All statistical analyses of data were executed using GraphPad Prism. The consequents were shown as mean ± standard deviation (SD) of assays performed three time. The Student’s t test


14

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was utilized to analyze statistical significance between the two groups, and multiple comparisons were compared by one-way ANOVA with or without Tukey-Kramer. In all situations, p 99% conservation, F404 showed > 98% conservation and H357 had > 92% conservation, indicating that D715-2441 might possess broad anti-IAV activities.

DISCUSSION In the present study,, we found that D715-2441 exhibited strong antiviral effects against against various influenza subtypes, including H1N1, H3N2, H5N1, H7N9, and the clinical


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isolate 690 (H3), and exhibited low toxicity against MDCK and A549 cells. In particular, D7152441 effectively inhibited oseltamivir-resistant influenza A (H1N1) virus with the H274Y NA mutation, and the timeliness of antiviral activity was effective up to 72 h. Additionally, D7152441 and zanamivir showed a cooperative antiviral activity against viral infection, which might be an effective option of the drug combination for chemoprophylaxis and therapy of influenza. Based on the above results, D715-2441 is a prospective candidate as anti-influenza virus agents. Hence, the mechanism of D715-2441 anti-influenza activity should be explored. The influenza virus life cycle includes viral entry, replication, and release. HA and NA is a glycoprotein found on the surface of influenza viruses, which mediates viral entry and promotes progeny virion release and spread, respectively. Our data showed that D715-2441 could not inhibit HA and NA activity. However, we found that D715-2441 strongly inhibited influenza viral replication after post-IAV infection. Generally, influenza virus takes 8-10 h to accomplish one life cycle 38. The results of different time points for the compound administrations provided evidenced that D715-2441 directly acted on the early step of the viral replication cycle of influenza virus. Therefore, we speculated that D715-2441 might target viral proteins that involved in the early stage in the viral replication cycle. Recently, Xofluza, the first PB2 cap-binding activity inhibitor, has been accredited and marketed in Japan as a broad-spectrum antiviral drug for influenza with a continuously effective period and high patient compliance

40

. Additionally, the PB2 subunit inhibitor drug pimodivir

(JNJ-63623872) has been reported and evaluated in a recent Phase IIa study 41-43, indicating that the cap-binding site in PB2 has become a focused target for antivirals. More recently, numerous efforts are underway to develop more promising PB2 cab-binding inhibitors to combat and


20

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seasonal influenza. In our effort to develop novel antivirals, by detection of luciferase expression in the 293T cells, we found that D715-2441 could diminish vRNP activity in a dose-dependent manner. The vRNP, including the viral RNA-dependent RNA polymerase (PA, PB2, and PB1) together with NP, mediates the viral transcription and replication occurred in the host cell nucleus

44

. Through a unique "cap-snatching" mechanism, PB2 subunit recognizes and binds to

the 5’-capped RNA fragments from host pre-mRNAs and complete the replication of viral RNA 22, 45

. Significantly, our data demonstrated that PB2 proteins accumulated and localized in both

the nucleus and cytoplasm of the infected-MDCK cells upon D715-2441 treatment at 24 h pi, in contrast to the control group. These results suggested that D715-2441 impaired polymerase activity and affected the nuclear transport of PB2 proteins, suggesting that the mechanism of D715-2441 against influenza virus infection might be associated with PB2 activity. The results of the SPR assay confirmed our deduction that D715-2441 and the influenza virus polymerase PB2 subunit had a specific interaction. Compared with the affinity of PB2cap proteins and PB219, D715-2441 had a significantly higher binding affinity to PB2cap proteins. Additionally, the FP assay showed that D715-2441 could dose dependently prevent PB2cap and m7GTP binding in a dose-dependent manner, further strengthening the result of the SPR experiment. Consequently, these findings validated that D715-2441 presented anti-influenza activity by binding the PB2cap and subsequently affecting polymerase activity, thereby hindering viral replication. The independently folded domain within PB2 subunit cap-binding domain (residues 318–483) is important for the initiation of viral mRNA transcription

18

. As reported previously, the cap

analog m7GTP could direct interact with PB2 cap via the conserved residues Phe323, Ser324, Arg355, Lys339, Glu341, His357, Phe363, Lys376, Phe404, and Asn429

35, 46

. Simultaneously,

D715-2441 bound to the same binding pocket of the PB2cap protein constituted by a β-sheet,


21


Page 22 of 39

and the flat condensed ring structure exactly matched the shape characteristics of the binding pocket. Specifically, D715-2441 formed hydrogen bonds with amino acid residues Glu361, Lys376, and Phe404 via the hydroxyl on the phenyl ring at 2.58 Å, 2.83 Å and 3.19 Å, respectively. These strong hydrogen bonds provided powerful electrostatic interactions (Grid_es = -7.584979 kcal/mol). Moreover, D715-2441 also formed sandwich and parallel-displaced π-π stacking interactions with His357, Phe404, and Phe323 through aromatic ring structures and hydrophobic interactions with amino acids, such as Phe404 and Phe325, which led to a powerful van der Waals interaction (Grid_vdw = -41.401646 kcal/mol). The van der Waals interactions were clearly the main driving force for the binding of this compound to PB2cap, and the electrostatic interaction played an important role in the binding orientation and enhancement. Accordingly, D715-2441 might exhibit inhibitory activity through the recognition and interaction of PB2 cap-binding pocket to destroy the polymerase activity of influenza A virus. Moreover, further python scripting showed that the five amino acids were conservative functional sites of PB2cap. By targeting these sites, D715-2441 might be applied to existing types of resistance. Additionally, considering its high price and further usage amount for animal experiments in vivo, D715-2441 was synthesized through protocols optimized from an earlier report and further purified to > 98% (Figure S5). Compared with the general procedure for its synthesis as described previously, we obtained a higher purity of D715-2441 by optimizing the synthesis conditions. In conclusion, the present study demonstrates that D715-2441 is a novel antiviral compound that interferes with the conserved cap-binding domain of the PB2 protein, resulting in the inhibition of influenza virus replication. Notably, these findings provide a new strategy for the design of broad-spectrum anti-influenza drugs that provide cross-subtype protection.


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Furthermore, considering the simple structure and high efficiency inhibition of influenza virus, D715-2441 is a prospective candidate for antivirals and can hopefully be developed as a lead compound to achieve a better effect class of influenza virus polymerase complex PB2 subunit inhibitor drugs.

AUTHOR INFORMATION Corresponding Author *

Tel:±8620-6164-8590. E-mail: [email protected]

*

Tel:±8620-6164-8590. Fax: +8620-6164-8655. E-mail: [email protected]

Author Contributions J Yang designed the research work, analyzed the data, and wrote the manuscript; T Liu performed the antiviral experiments; MM Liu performed SPR experiment. FM Chen performed molecular docking study. FZ Chen and Q Huang contributed to synthesize and purify the compounds. YX Tian analyzed the conservation of binding sites. SW Liu helped in project design and guide and revised the manuscript.

ACKNOWLEDGMENT This work was financially supported by the Natural Science Foundation of Guangdong Province (No. 2016A030313591) and the Pearl River S & T Nova program of Guangzhou (No. 2014J2200033). CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.


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TABLES Table 1. Inhibitory activities of D715-2441 against infection by multiple subtypes of influenza A viruses. Inhibition activity of D715-2441а Virus strain

SI IC50 (µM)

CC50 (µM) (CC50/IC50)

A/Puerto Rico/8/34 (PR8; H1N1)

1.702±0.021

>200

>117.5

A/FM-1/1/47 (H1N1)

3.594±1.251

>200

>55.6

A/Aichi/2/68 (H3N2)

4.300±1.103

>200

>46.5

A/Vietnam/1194/2004 (H5N1)

3.79±0.487

>200

>52.8

A/Anhui/1/2013 (H7N9)

3.19±0.298

>200

>62.7

the influenza A viruses 690 (H3)

4.439±0.472

>200

>45.1

Oseltamivir-resistant influenza A (H1N1) viruses with NA-H274Y 3.361±0.416 mutation a

>59.5 >200

The samples were examined in MDCK cells in triplicate. Data was depicted as mean±S.D.

IC50: 50% inhibitory concentration. CC50: 50% cellular cytotoxicity concentration.


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Table 2. Effect of combinations of D715-2441 with zanamivir. IC50 equivalentа

Combination tested

а

D715-2441

zanamivir

FICIb

7:1

1.48

0.21

1.69

3:1

0.60

0.20

0.80

2:1

0.68

0.34

1.02

1:1

0.33

0.33

0.66

1:3

0.10

0.30

0.40c

1:7

0.08

0.56

0.64

The concentrations of D715-2441 or zanamivir was set to its IC50 values.

b

c

D715-2441:zanamivir

FICI was the summation of each combination of D715-2441 and zanamivir IC50 equivalent.

A remarkable cooperative effect is detected when FICI value < 0.5.


29


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Table 3. Analysis of amino acid conservation of D715-2441 binding sites.

a

Binding site residues

Position

Mutant population

(conservation ratio %)а

H

357

65

92.81

E

361

1

99.89

F

323

2

99.78

F

404

11

98.78

K

376

1

99.89

The conservation ratio of the five predicted amino acids was analyzed by the Python script.

PB2cap sequences were obtained from the influenza virus resource at NCBI.


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FIGURES A HO

OH

O O

B

C

Figure 1. D715-2441 inhibited influenza A virus infection. (A) Chemical structure of D7152441. (B) D715-2441 protection against influenza viral infection in PR8-virus infected MDCK cells (MOI = 0.01). Images of the cells were acquired at ×10 magnification at 48 h pi. (C) The D715-2441 could dose-dependently reduce viral plaque formation in PR8-virus infected MDCK cells.


31


A

Page 32 of 39

B

C

D

Figure 2. D715-2441 exhibited dose-dependent inhibition of viral replication in IAV-infected cells. The PR8-virus infected MDCK cells (MOI=0.01) were intervened with serially diluted D715-2441

for

24

h.

(A)

Monitoring

of

influenza virus

production

by

indirect

immunofluorescence microscopy. The green foci represent viral NP proteins (left column).


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Nuclei were counterstained with DAPI (blue). The protein and DAPI images were merged (right column). Original magnification, 10×. (B) The production of viral NP protein was examined using western blotting. (C) Viral mRNA expression was detected using RT-PCR. (D) Viral mRNA expression was measured using RT-PCR at the indicated time. Ribavirin was used as a control. Results are shown as the mean ± SD. (NS, not significant, *p < 0.05 and **p < 0.01).


33


A

Page 34 of 39

C

B

Figure 3. Inhibition of IAV replication by D715-2441 was detected using time-of-addition assay. (A) Anti-influenza virus effects of D715-2441 under different treatments. D715-2441 coincubated with influenza PR8 virus for 30 min at 37 °C before adding on MDCK cell monolayers in pre-infection treatment. The virus-infected MDCK cells were intervened with D715-2441 in post-infection treatment. Entire infection refers to removal of the mixture and D715-2441 and subsequently culture cells with a medium containing the compound. Then, culture supernatants of each group (i.e., pre-infection group, post-infection group, and entire infection group) were collected and the antiviral effect of D715-2441infection was determined at 48 h pi as described in Material and Methods. (B) D715-2441 was added to MDCK cells at different intervals after PR8-virus infection. The production of the viral NP protein was detected by western blotting. (C) The viral mRNA expression was detected using RT-PCR at 10 h pi. The


34

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results represent the average of three-time experiments. Data are presented as mean ± SD. (NS, not significant, **p < 0.01 and ***p < 0.001).

A

B

Figure 4. D715-2441 affected influenza viral polymerase activity and localized PB2 protein in the nuclei and cytoplasm. (A) The effect of D715-2441 on viral polymerase activity was assessed by a luciferase-based minireplicon assay. Zanamivir was used as a negative control. The results represent the average of three-time experiments and data are displayed as mean ± SD. (NS, not significant, and *p < 0.05). (B) Virus-infected MDCK cells were intervened with D715-2441 and fixed at 24 h pi. The green foci represent viral PB2 proteins (left column). Nuclei were counterstained with DAPI (blue). The protein and DAPI images were merged (right column). Original magnification, 63×.


35


A

Page 36 of 39

B

Figure 5. D715-2441 showed a specific interaction with the viral PB2cap protein. (A) The binding affinity of D715-2441 to viral PB2cap protein was obtained from SPR measurements. Influenza PB2cap proteins were fixed on a sensor chip. Next, D715-2441 or PB2-19 at different concentrations was injected over the chip surface with 300 s of the contact time and dissociation time. (B) The specific binding between PB2cap protein and D715-2441 was detected using the FP assay. A mixture of PB2cap (1 µM) and FITC-m7GTP (20 nM) was added to serially diluted D715-2441 for 30 min. Subsequently, fluorescence polarization was measured. Data are expressed as the mean ± SD.


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A

Figure 6. Molecular docking analysis. In the three-dimensional structural analysis, D715-2441 is presented as colored stick models, while the residues interacting with PB2cap protein are marked with gray. D715-2441 was predicted to interact with PB2cap via H357, F323, F404, E361, and K376 with π-π stacking, hydrophobic interactions or hydrogen bonds. H357, F323, F404, E361, and K376 were acquired from IAV resource at NCBI and analyzed for conservation. The five predicted amino acids were derived from influenza virus strains from various sources, including human, swine and avian-origin IAVs.


37


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SUPPORTING INFORMATION In supporting information, we have provided the cytotoxicity of D715-2441 against MDCK and A549 cells and found it exhibited a low cytotoxic effect at concentrations up to 200 µM. In order to confirm the non-cell strain dependence of research results, we have detected the impact of D715-2441 on the replication of IAVs in A549 cells. Additionally, the results were provided about the influence of D715-2441 on pseudovirus infection and NA activity. It displayed that D715-2441 was unable to inhibit both viral entry and release. PB2-19, a PB2cap inhibitor, was measured the binding affinity to influenza PB2cap protein as a positive control. Finally, we have listed the synthesis, the 1H-NMR spectra, 13C NMR spectra and HRMS data of D715-2441.

LIST OF SUPPORTING INFORMATION Figure

Experiment contents

Figure S1

The effect of D715-2441 on cell viability. The impact of D715-2441 on the replication of influenza A viruses in

Figure S2 A549 cells. Figure S3

Influence of D715-2441 on pseudovirus infection and NA activity.

Figure S4

PB2-19 binding affinity to influenza PB2cap protein. The synthesis, the 1H-NMR spectra, 13C NMR spectra and HRMS data

Figure S5 of D715-2441.


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Title: A small-molecule compound has anti-influenza A virus activity by acting as a ‘‘PB2 inhibitor” Authors: Teng Liu, Miaomiao Liu, Feimin Chen, Fangzhao Chen, Yuanxin Tian, Qi Huang, Shuwen Liu*, Jie Yang*


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PB2 inhibitor - ACS Publications

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