Article Cite This: J. Nat. Prod. 2018, 81, 2722−2730
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Spirostaphylotrichin X from a Marine-Derived Fungus as an Antiinfluenza Agent Targeting RNA Polymerase PB2 Jianjiao Wang,†,§,# Feimin Chen,‡,# Yunhao Liu,†,▽ Yuxuan Liu,†,▽ Kunlong Li,† Xiliang Yang,⊥ Shuwen Liu,*,‡,∥ Xuefeng Zhou,*,†,∥ and Jie Yang*,‡
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†
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China ‡ 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, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ⊥ Department of Pharmacy, Medical College, Wuhan University of Science and Technology, Wuhan 430065, People’s Republic of China ∥ State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, People’s Republic of China S Supporting Information *
ABSTRACT: A new spirocyclic γ-lactam, named spirostaphylotrichin X (1), and three related known spirostaphylotrichins (2−4) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures were determined by spectroscopic analyses. Spirostaphylotrichin X (1) displayed obvious inhibitory activities against multiple influenza virus strains, with IC50 values from 1.2 to 5.5 μM. Investigation of the mechanism showed that 1 inhibited viral polymerase activity and interfered with the production of progeny viral RNA. Homogeneous time-resolved fluorescence, surface plasmon resonance assays, and a molecular docking study revealed that 1 could inhibit polymerase PB2 protein activity by binding to the highly conserved region of the cap-binding domain of PB2. These results suggest that 1 inhibits the replication of influenza A virus by interfering with the activity of PB2 protein and that 1 represents a new type of potential lead compound for the development of anti-influenza therapeutics.
I
nidase (NA) inhibitors, which are used for prophylaxis and early treatment of influenza virus infection.10 However, M2 ion channel inhibitors are not recommended currently to defend against influenza due to widespread resistance and potential central neurotoxicity.11 NA inhibitors are the front-line antiinfluenza drugs in current clinical use, such as zanamivir, peramivir, and oseltamivir.12 Although typically not associated with fatalities anymore, increased drug resistance still challenges the effectiveness of established NA inhibitors.13−15 Thus, development of new antiviral agents with new modes of action should be intensified. Influenza polymerase may be an ideal anti-influenza drug target, and a number of viral polymerase inhibitors have been reported. As a successful example, baloxavir marboxil (Xofluza; baloxavir) has been licensed for use for the treatment of both influenza A and B virus infections in Japan. Baloxavir marboxil is a new capdependent endonuclease inhibitor, which blocks the proliferation of influenza virus by inhibiting the initiation of mRNA
nfluenza virus is a segmented, negative-sense, single-strand RNA virus belonging to the Orthomyxoviridae family,1 and influenza A virus (IAV) is the most widely spread of types A− D influenza viruses.2 The outbreak of IAV H1N1 in Spain caused an estimated 500 million human infections and killed around 20−50 million people worldwide in 1918.3 There have been several other influenza pandemics since 1918, and their high morbidity and mortality caused serious social and economic problems.4,5 Influenza virus outbreaks still occur today, and new emerging influenza viruses have been identified, such as H7N9, H5N1, and H5N8, which has led to increased worldwide attention.6,7 Influenza vaccine is the primary method used to prevent influenza outbreaks. However, inactivated influenza vaccine and live attenuated vaccine are prone to lose activity due to reduced biological efficiency and immune system evasion caused by antigenic drift and shift.8,9 Therefore, vaccines can be powerless to help counteract circulating strains with evolutionary drift. Antiviral drugs might be a good treatment option to prevent viral infection, and there are two classes of anti-influenza drugs, M2 ion channel inhibitors and neurami© 2018 American Chemical Society and American Society of Pharmacognosy
Received: August 6, 2018 Published: December 5, 2018 2722
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
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synthesis.16 Another novel anti-influenza inhibitor, pimodivir, also known as VX-787, possesses broad-spectrum antiinfluenza activity by disrupting PB2 cap-snatching activity.17 Marine alga-derived endophytic fungi have been reported as an important resource of bioactive and medicinal compounds.18,19 During our chemical studies of marine-derived fungi, three new eremophilane sesquiterpenes, dendryphiellins H−J, and three new phthalide natural products were obtained from the marine alga derived fungus Cochliobolus lunatus SCSIO41401, fermented without sea salt.20 In order to discover more diverse and bioactive compounds from this strain, a fermentation supplemented with 20 g/L sea salt was recently undertaken, and four spirostaphylotrichin compounds were obtained from this strain for the first time. Spirostaphylotrichin or triticone analogues, which are spirocyclic γ-lactams, are mainly produced by several endophytic fungal strains of Curvularia,21 Pyrenophora,22−24 and Staphylotrichum.25 Many of the spirostaphylotrichins are well known as phytotoxins and have the potential to be used as herbicides or biocontrol agents.22,23 However, the medicinal potential of these compounds has not been reported. Described herein are the isolation, structure determination, and biological evaluation of the four spirostaphylotrichins from the marine-derived fungus C. lunatus SCSIO41401. The new compound spirostaphylotrichin X (1) displayed anti-IAV activity, and its mode of action was also explored.
Table 1. NMR Data for 1 (CD3OD, 1H 500 MHz, 13C 125 MHz)
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RESULTS AND DISCUSSION A 50 L fermentation of C. lunatus SCSIO41401 was extracted with EtOAc, and the resulting extract was separated by chromatographic methods to yield compounds 1 and 2 and a mixture of 3 and 4.
position
δC, type
1 3 4 5 6 7 8 9 10 11 12 13 14 15 16
169.3, C 97.0, C 68.4, CH 59.3, C 75.0, CH 199.0, C 122.8, CH 154.1, CH 131.3, C 18.0, CH3 149.7, CH 24.7, CH2 13.6, CH3 64.8, CH3 51.1, CH3
δH (J in Hz)
4.48, s 4.74, s 5.83, d (9.9) 7.09, d (9.9) 1.38, 6.10, 2.15, 1.04, 3.95, 3.39,
s t (8.0) m; 2.02, m t (7.4) s, CH3 s
Figure 1. Key HMBC, COSY, and NOESY correlations of 1.
the H-4/H3-11 NOESY correlations in the reported data of spirostaphylotrichins W22 and V21 with a cis configuration of C-4 and C-3. The absence of a NOESY correlation between H4 and H2-13 indicated that the OH-4 was located on the same face of the lactam ring as the C-13 methylene protons.23 The relative configuration of C-6 was also confirmed by the absence of an NOESY correlation of H-6 with H3-16 or H3-11.23 So, the relative configuration of 1 was determined as 3S*,4R*,5S*,6S* by NOESY correlations mainly (Figure 1). The electronic circular dichroism (ECD) spectrum of 1 showed positive Cotton effects (CEs) at about 241 and 332 nm and a negative CE at about 288 nm (Figure S4, SI), which were identical to those of spirostaphylotrichins with the absolute configuration 4R, 5S, 6S.25 Therefore, the structure of 1, the C-3 epimer of staphlotrichin V and the C-4 epimer of staphylotrichin W, was finally determined as (3S,4R,5S,6S,8Z,10Z)-4,6-dihydroxy-2,3-dimethoxy-3-methyl10-propyliden-2-azaspiro[4.5]dec-8-ene-1,7-dione. Compound 2 was characterized as spirostaphylotrichin A25 (or triticone C23), by comparison of its NMR and MS data with literature data. Spirostaphylotrichin R (or triticone F, 3) and triticone E (4), with the only difference being the C-6 configuration, were obtained as a mixture. Compounds 3 and 4 were difficult to isolate, and the mixture, about a 5:3 ratio indicated by a 1H NMR spectrum, was thermally stable as reported.23 The 1H and 13C NMR data (Tables S1, S2) were assigned and the structures were determined by comparison with literature data of the same mixture.23 The only differences between 1 and 3 are a methoxy at C-3 in 1 as compared to a hydroxy in 3 and the C-3 configuration. Because C-3 is a masked carbonyl, and MeOH was used in several steps of the isolation procedure, 1 could be an artifact
Compound 1 was isolated as an amorphous solid. Its molecular formula was established by HR-ESIMS to be C15H21NO6. The 1H NMR spectrum of 1 displayed signals attributed to two methoxy groups at δH 3.95 (s, H3-15) and 3.39 (s, H3-16); two other methyls at 1.38 (s, H3-11) and 1.04 (t, J = 7.4 Hz, H3-14); one methylene at δH 2.15 (m, H2-13a) and 2.02 (m, H2-13b); three olefinic or aromatic protons at δH 7.09 (d, J = 9.9 Hz, H-9), 6.10 (1H, t, J = 8.0 Hz, H-12), and 5.83 (d, J = 9.9 Hz, H-8); and two oxygenated methines at δH 4.74 (s, H-6) and 4.48 (s, H-4). The 1H and 13C NMR spectra of 1 were similar to those of reported spirostaphylotrichins W and V (Tables 1, S1, and S2),22 except for some small differences with those chemical shifts of C-3, C-4, H-4, and H11. The main HMBC and COSY correlations (Figure 1) of 1 suggested it has the same planar structure as spirostaphylotrichins W and V, obtained from different fungal sources. The Z-configurations of the C-8/C-9 and C-10/C-12 double bonds were determined by the NOESY correlations observed between H-8/H-9 and H-12/H-9, respectively (Figure 1). The NOESY correlation between H-4 and H3-16 indicated the trans configuration of C-4 and C-3 in 1 when compared with 2723
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
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Figure 2. Cytotoxic and antiviral activities of 1. (A) Effect of 1 on the cell viability in MDCK cells. (B) Compound 1 reduced A/Puerto Rico/8/34 (H1N1) viral plaque formation in MDCK cells in a dose-dependent manner.
formed during processing of the extract. Based on the reported biogenetic interrelationships of the spirostaphylotrichins, compounds 1 and 3 are likely to be derived from 2.26 Weak or Inactive Germination Inhibitory, Cytotoxic, and Antibacterial Activities of the Spirostaphylotrichins. Several spirostaphylotrichins were reported with phytotoxic or herbicidal activities. In the reported phytotoxic bioassays against cheatgrass (Bromus tectorum) coleoptiles, spirostaphylotrichin A (2) showed activity (10−3 M), while the mixture of spirostaphylotrichin R (3) and triticone E (4) was inactive.22 In our germination inhibition assay, 1, 2, and the mixture of 3 and 4 (about a 5:3 ratio) did not arrest the germination of Bermuda grass (Cynodon dactylon) and Arabidopsis thaliana seeds. They were also inactive in the cytotoxicity tests (IC50 > 50 μM) against three renal cancer cell lines (ACHN, 786-O, and OS-RC-2), a human liver cancer cell line (HepG2), and a human gastric cancer cell line (SGC7901) and in antibacterial tests (MIC > 100 μg/mL) against four bacterial species (Escherichia coli, Staphylococcus aureus subsp. aureus Rosenbach, Erysipelothrix rhusiopathiae, Pasteurella multocida subsp. Multocida). Compound 1 Inhibits Different Subtypes of IAV Infection. Compound 1 exhibited low cytotoxic effects against the target cells, Madin-Darby canine kidney (MDCK) cells, even at concentrations up to 200 μM (Figure 2A). On the basis of the results from an MTT-based cytopathic effect (CPE) reduction assay and microscopy, 1 showed broadspectrum inhibitory effects against different subtypes of IAV infection, including A/Aichi/2/68 (H3N2), A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 (H1N1), an oseltamivirresistant strain with an NA-H274Y mutation, and a clinical isolate 690 (H3 subtype) strain, with IC50 values ranging from 1.2 to 5.5 μM (Table 2).27 A plaque reduction assay against the A/Puerto Rico/8/34 (H1N1) virus was used to identify and confirm antiviral activity of 1 (Figure 2B). Compound 1 Inhibits IAV Replication. The impact of 1 on viral replication was assessed by indirect immunofluorescence. MDCK cells were infected with influenza PR8 virus at a multiplicity of infection (MOI) of 1.0, followed by being treated with 1 at the indicated concentrations. As shown in Figure 3A, obvious alterations in the compound-treated cells were observed at 24 h postinfection. Also, levels of both viral RNA and protein in the virus-infected cells were markedly decreased (Figure 3B,C). Taken together, these results showed that 1 inhibited influenza A virus replication in a dosedependent manner.
Table 2. Inhibition Activity of 1 against Various IAV Strains Iinhibition activity of 1a IAV strain A/Aichi/2/68 (H3N2) A/FM-1/1/47 (H1N1) A/Puerto Rico/8/34 (H1N1)b A/PR/8/34 (H1N1) with NA-H274Y influenza A 690 (H3)
IC50 (μM)
CC50 (μM)
± ± ± ± ±
>200 >200 >200 >200 >200
4.1 5.5 1.6 1.2 1.7
0.5 2.3 0.6 0.5 0.6
a Tested in triplicate, mean ± SD. IC50, the 50% inhibitory concentration ; CC50, cytotoxic concentrations. bCompound 2 showed weak inhibition activity with an IC50 value of 21 ± 6.5 μM against A/Puerto Rico/8/34 (H1N1). Activity was not detected with the mixture of 3 and 4 at a concentration of 200 μM.
Compound 1 Interferes with the Early Phase of the Viral Replication Cycle. The influenza virus life cycle can be divided into three main stages: virus entry, replication, and release, respectively. Here, a time-of-addition assay was used to explore when 1 acts in the viral life cycle. The results indicated that 1 acts during the postinfection process to inhibit virus replication (Figure 4A), rather than having an effect on virus entry or release of progeny virus. Furthermore, pseudovirus neutralization, hemagglutinin (HA) inhibition, and neuraminidase (NA) inhibition assays were carried out, indicating that compound 1 did not affect the function of influenza virus membrane proteins HA and NA (Figure S14). The entire viral replication cycle requires approximately 10 h, including early and late steps of viral replication. As shown in Figure 4B, the compound was added to the infected cells at defined time points, which covered the first viral replication cycle. After incubation for 10 h postinfection, the levels of nucleoprotein (NP) and HA mRNA were measured by Western blotting and quantitative RT-PCR, respectively. The reduction of both viral NP production and HA mRNA levels suggested that 1 interferes with an early viral replication step (Figure 4C,D). Compound 1 Exerts Antiviral Activity by Affecting the PB2 Protein. In the early stages of influenza virus infection, the virus is mainly shelled and releases the viral ribonucleoprotein complex (vRNP), which is the smallest functional unit of influenza virus replication.28 The released vRNP enters the nucleus and begins the progeny virus replication. Here, a virus-inducible Fluc reporter gene system was used to assess viral vRNP activity. The results demonstrated that 1 could inhibit the activity of vRNP in 293T cells infected with the tested virus at different MOI (Figure 5A). Influenza virus RNA-dependent RNA polymerase 2724
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
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Figure 3. Inhibitory effect of 1 on viral replication in MDCK cells. (A) Effect of 1 on the expression of viral NP protein detected by immunofluorescence microscopy. (B) Effect of 1 on the expression of viral HA mRNA detected by quantitative RT-PCR. The samples were tested in triplicate, and the data are presented as means ± SD; *p < 0.05. (C) Effect of 1 on the expression of viral NP protein detected by Western blotting at the indicated postinfection time points.
Figure 4. Compound 1 interfered with the early phase of the viral replication cycle. (A) Inhibitory effect of 1 on viral infection observed under three methods of treatment, named preinfection, postinfection, and entire infection. (B) Exposure times of 1 during a single cycle of influenza virus replication. (C) Production of viral NP protein during a single cycle of influenza virus replication. (D) Expression of viral NP mRNA during a single cycle of influenza virus replication. The data represent the average of three independent experiments and are shown as the mean ± SD (**p < 0.01, ***p < 0.001).
is a central component of the vRNP and is involved in the transcription of viruses and the formation of progeny viral nucleic acids. To investigate whether 1 affected RNA polymerase activity, we extracted total intracellular RNA at 3
and 6 h after virus infection and reverse-transcribed it into vRNA by using specific primers. The results of real-time quantitative PCR assays revealed that 1 inhibited the production of vRNA, whereas penamivir, a negative control 2725
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
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Figure 5. Compound 1 exerted antiviral activity by affecting the polymerase PB2 cap-binding domain. (A) Inhibitory effect of 1 on vRNP activity evaluated using a vRNP activity inhibition assay. (B) Inhibitory effect of 1 on the replication of intracellular virus-specific vRNA. (C) Experiment diagram for the HTRF assay. (D) Specific binding between the PB2-cap protein and 1 detected using the HTRF assay. (E) Affinity of 1 for PB2cap analyzed using an SPR assay. (F) Molecular docking of 1 with PB2-cap. Data represent the average of three independent experiments and are shown as the mean ± SD (ns, nonsignificance, and *p < 0.05, **p < 0.01, ***p < 0.001).
fluorescence intensity. As the results show, 1 inhibited m7GTP and viral PB2-cap binding dose-dependently (Figure 5D). In addition, the surface plasmon resonance (SPR) analysis provided affinity data and indicated 1 strongly bound to the PB2-cap protein with a KD value of 77 nM (Figure 5E), compared with the positive control compound (D715-2441) of 3.6 μM (Figure S15A). Taken together, these data clearly demonstrate that 1 directly targets the influenza A virus PB2cap as a cap-binding competitor. Molecular Docking of 1 with PB2-Cap. We next investigated the possible binding sites on the PB2 protein via molecular docking. Based on the 3D molecular structure of 1 and the crystal structure of PB2-cap from PDB 4CB4, we conducted docking studies and obtained four potential conformers by using Dock 6.7 molecular modeling simulation software. The results for the optimal conformations demonstrated that 1 could bind within the cap-binding pocket of PB2, a pocket previously reported.33−35 As shown in Figure 5F, hydrogen bonds and hydrophobic interactions were formed
drug, did not (Figure 5B). Therefore, we propose the activity of 1 is via inhibition of the influenza virus RNA polymerase. Influenza RNA polymerase is heterotrimeric, formed by subunits PB1, PB2, and PA. The polymerase PB2 subunit is understood to initiate viral transcription via a unique mechanism named “cap-snatching”.29,30 In this process, the cap-binding domain of the PB2 subunit, also known as PB2cap, binds to the 5′-cap of the host pre-mRNA, followed by cleavage of 10−13 nucleotides dominated by PA endonuclease.31,32 Therefore, the PB2-cap is a key protein of influenza polymerase and is indispensable in viral transcription, implying that it might be a promising target for antiviral drugs. A homogeneous time-resolved fluorescence (HTRF) assay was used to detect the interaction between 1 and the influenza PB2-cap protein. As shown in a diagram of the experiment (Figure 5C), when the compound specifically disrupts the binding between PB2-cap and the cap analogue m7GTP, the donor energy cannot be completely transferred to the fluorescent receptor, resulting in a decrease in the relative 2726
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
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cultures from 200 flasks (50 L total) were harvested. Both the broth and mycelia were extracted with EtOAc for further chemical study. Isolation and Purification. The EtOAc extract (51 g) was separated by silica gel column chromatography (CC) to yield seven fractions (fr.a−fr.g), with a gradient of petroleum ether/EtOAc (50:0, 50:1, 20:1, 5:1, 2:1, 1:1, and 0:1). In fraction fr.c, several compounds that differed compared with those we previously reported in this strain were observed. Fraction fr.c was chromatographed over Sephadex LH20 (CHCl3/MeOH, 1:1), followed by repeated silica gel CC (CHCl3/ MeOH, gradient elution 200:1−20:1) purification, to afford compounds 1 (16.2 mg), 2 (6.6 mg), and the mixture (5.2 mg, about a 5:3 ratio) of 3 and 4. Compounds 3 and 4 could not be isolated from each other by repeated silica gel CC and semipreparative HPLC (25% to 40% MeCN/H2O, 2 mL/min). Spirostaphylotrichin X (1): white, amorphous solid; [α]20D +6.2 (c 0.15, CHCl3); UV (CHCl3) λmax (log ε) 289 (0.83) nm; ECD (c 0.15, CHCl3) λmax (Δε) 241 (+ 0.54), 288 (− 2.40), 332 (+ 0.48) nm; IR νmax 3401, 2970, 1717, 1668, 1616, 1283, 1138, 1107, 851 cm−1; 1H and 13C NMR data, Table 1; HR-ESIMS m/z 334.1267 [M + Na]+ (calcd for C15H21NO6Na, 334.1261). Germination Inhibition, Cytotoxicity, and Antibacterial Assays. The germination inhibition assay of seeds (Cynodon dactylon and Arabidopsis thaliana) was carried out as previously described.36 Briefly, 10 surface-sterilized seeds were added to each well of a sterile tissue-culture plate, and 100 μL of the test solution (1, 2, and the mixtures of 3/4, 100 μg/mL with 5% EtOH) was added to the agar medium in each of three wells. After being incubated at 20 °C with a 12:12 h photoperiod for 7 days, seed germination was scored as reported. The cytotoxicity (MTT assay; ACHN, 786-O, OS-RC-2, HepG2, SGC7901, and also MDCK cells) and antibacterial assays, against E. coli (ATCC25922), S. aureus subsp. aureus Rosenbach (ATCC25923), E. rhusiopathiae (ATCC19414), and P. multocida subsp. Multocida (ATCC43137), purchased from the American Type Culture Collection (ATCC), were carried out as described in our recently published paper.20 Viruses and Plasmids. MDCK and human embryonic kidney (293T) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin (PS) and 10% fetal bovine serum (FBS). The subtypes of IAV included A/Aichi/2/68 (H3N2), A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 (H1N1), A/ PR/8/34 (H1N1) with NA-H274Y, and the influenza A viruses 690 (H3) and were propagated from 8-day-old chicken embryos. The viruses were stored at −80 °C after being separated. The HIV backbone plasmid (pNL4-3.luc.R-E-), A/Thailand/Kan353/2004HA, and A/Thailand/Kan353/2004-NA were supplied by Professor Frank Kirchhoff (University Ulm, BW, Germany). The pPolI-Fluc (firefly luciferase reporter plasmid) was kindly provided by Professor Bojian Zheng (University of Hong Kong, Hong Kong, China). Plaque Assay. The monolayer MDCK cells in six-well plates were infected with A/Puerto Rico/8/34 (H1N1) virus at an MOI of 0.005 at 37 °C for 1 h. Subsequently, the culture supernatant was discarded and 3 mL of serum-free MEM containing serially diluted 1, 2% microcrystalline cellulose, and 1.5 μg/mL trypsin was added to each well. After 72 h of infection, semisolid medium was replaced by 1 mL of staining solution with 4% paraformaldehyde and 2% crystal violet for 20 min as previously described.37 The amount of plaque formation reflects the inhibitory effect of 1 on the tested virus. Immunofluorescence Microscopy. Immunofluorescence microscopy was conducted as previously reported.38 The MDCK cells were infected with the virus at an MOI of 0.01 for 1 h, followed by inoculation with increasing concentrations of 1 for an additional 24 h. Afterward, the cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 min, and then covered with 3% bovine serum albumin (BSA) for 1 h at room temperature. The cells were then incubated with anti-NP antibody (1:200 dilution, GenTax) overnight at 4 °C and with a fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:200 dilution, Santa Cruz) at room temperature for 4 h. 4,6-Diamidino-2-phenylindole (DAPI) staining was used as a nuclear marker. The fluorescence was visualized by a fluorescence microscope (Nikon).
from 1 to the key residues Arg 355, Asn 429, Met 431, Phe 325, and Lys 339 of PB2. This is the first discovery of anti-IAV activity for a spirostaphylotrichin, a fungus-derived spirocyclic γ-lactam structure. Impressively, the new compound spirostaphylotrichin X (1), with weak cytotoxicity to a virus-susceptible cell line, showed obvious and broad anti-IAV activity in vitro, including against a clinical isolate and an oseltamivir-resistant influenza A (H1N1) virus. Also, viral protein production and gene expression were markedly decreased in the compoundtreated cells. Together, these experiments demonstrate that 1 could be a promising candidate as an antiviral agent and prompted us to explore the mechanisms of the anti-IAV activity of 1. Mechanistic studies showed that 1 acts in a postinfection process and in the early phase of viral replication. A follow-up study indicated 1 could significantly inhibit vRNP activity. An HTRF assay, an SPR experiment, and molecular docking studies revealed that 1 could target the polymerase PB2 protein by binding to the highly conserved region of the cap-binding domain of PB2. Therefore, it is suggested that 1 inhibits the replication of IAV by interfering with the activity of the PB2 protein. As a crucial component of influenza virus RNA polymerase, the PB2 subunit binds to the host RNA cap and supports the function of the PA subunit to “snatch” the cap from host premRNAs as previously described.31 More importantly, the capbinding domain of the PB2 subunit is fairly conserved among different subtypes of influenza virus and might be able to provide a relatively high genetic barrier to drug resistance. Clearly, the PB2 subunit has become an attractive target for the development of antiviral drugs. Our findings indicate that spirostaphylotrichin X (1) acts as a novel inhibitor of influenza virus replication, apparently by binding to critical residues in the functional region of the influenza A polymerase PB2 subunit, blocking viral cap-snatching. To summarize, our present study discovered a new spirostaphylotrichin compound from the marine-derived fungus C. lunatus SCSIO41401, with broad anti-IAV activities. A mode of action study showed that 1 inhibits the replication of IAV by targeting the viral polymerase PB2. This study provides a new type of potential lead compound for the development of anti-influenza therapeutics.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were performed on a PerkinElmer 341 polarimeter. UV and ECD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu). NMR spectra were obtained on Bruker AVANCE-500 or Bruker AC 700 MHz spectrometers with tetramethylsilane as an internal standard. HRESIMS data were recorded with a Bruker maXis Q-TOF in positive/negative ion mode. Column chromatography was performed on silica gel (Cosmosil) and Sephadex LH-20 (Amersham Biosciences). Semipreparative HPLC (Agilent, 1260 infinity) was performed using an ODS column (YMCpack ODS-A, 10 × 250 mm, 5 μm). Fungal Material and Fermentation. The fungal strain, identified as Cochliobolus lunatus SCSIO41401, was isolated from a marine alga collected in Yongxing Island, South China Sea, and deposited in the Center for Marine Microbiology, South China Sea Institute of Oceanology, CAS, China.20 The fermentation of the strain was identical to that of our recently published paper,20 except for adding sea salt (20 g/L) in the seed and production media. After incubation at 28 °C statically for 7 days, 2727
DOI: 10.1021/acs.jnatprod.8b00656 J. Nat. Prod. 2018, 81, 2722−2730
Journal of Natural Products
Article
Western Blotting. The monolayer MDCK cells were infected with influenza PR8 virus at an MOI of 0.01 for 1 h at 37 °C, followed by treatment with serial dilutions of 1. At 24 h postinfection, the cells were lysed and the protein concentrations were determined. Subsequently, the samples containing equal amounts of protein were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. The viral proteins were detected with anti-NP antibody (1:1000 dilution, Abcam), followed by anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000 dilution, Cell Signaling Technology). The loading control used a β-actin antibody (1:1000 dilution, Cell Signaling Technology) and a secondary antibody as mentioned above. The images were analyzed by ECL chemiluminescence exposure equipment. Quantitative Real-Time PCR. The expression of viral mRNA was detected using quantitative RT-PCR according to the previous description.37 In brief, the infected MDCK cells were treated with a series of concentration gradients of 1 for 24 h. Subsequently, total RNA was extracted by TRIzol reagent (Invitrogen) and quantified. The expression of the target gene was detected by quantitative RTPCR after being reverse-transcribed into cDNA and normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference gene. The expression of the target gene was shown in a classical 2−ΔΔCT method using 7500 software. Quantitative RT-PCR was also used to detect the expression of the intracellular viral RNA (vRNA) at various time points postinfection, with peramivir as a negative control.39 Briefly, the infected MDCK cells were treated with 1 at the indicated concentrations for 24 h. The total RNA was extracted and reverse-transcribed into cDNA at 3 and 6 h postinfection using the specific primers of the target genes. GAPDH was used as the internal control for cellular RNA.39 Subsequently, cDNA was used for quantitative RT-PCR as described above. The primers for quantitative RT-PCR are listed in Table S3. Time-of-Addition Assay. Three different treatments were used to observe the period of influenza virus life cycle affected by 1.38 To explore the effect of 1 on influenza virus entry, influenza PR8 virus at an MOI for 0.01 was inoculated with 1 for 30 min at 37 °C prior to addition of MDCK cells. After an additional 30 min at 37 °C, the culture supernatant was replaced by fresh medium containing 1 μg/ mL TPCK-trypsin, which was called the preinfection treatment. To examine the effect of 1 on influenza virus replication, the infected MDCK cells were inoculated with increasing concentrations of 1. After 1 h of incubation at 37 °C, the medium was replaced with fresh medium, which was called the postinfection treatment. For the entire infection treatment, the cells were infected with the mixture of virus and compound as described for the preinfection treatment. After removing the unabsorbed viruses, fresh medium containing 1 μg/mL TPCK-trypsin together with 1 was added to each well. The cells were cultured for another 48 h and the MTT-based assay described above was used to detect cell viability. vRNP Activity Inhibition Assay. The vRNP activity inhibition assay was performed as previously described with minor modifications.40 Briefly, 293T cells grown in 24-well plates were transfected with 50 ng of pPolI-Fluc, a firefly luciferase reporter plasmid, using Lipofectamine 3000 according to the manufacturer’s instructions. After 5 h of transfection, the cells were infected with different titers of PR8 influenza virus for 1 h. Subsequently, the supernatant was displaced by DMEM with 10% FBS containing 10 μM 1 or 0.1% DMSO. After 24 h, the cells were lysed and the luciferase reporter gene activity was measured and quantitated. Pseudovirus Neutralization Assay. The H5N1 pseudovirus was composed of a core protein from HIV and envelope proteins of HA and NA from influenza A/Thailand/Kan353/2004 (H5N1) as described previously.41 Briefly, 6 μg of HIV core plasmids, 2 μg of A/Thailand/Kan353/2004-HA, and 2 μg of A/Thailand/Kan353/ 2004-NA were transfected into 293T cells using Lipofectamine 3000 (Invitrogen). The pseudovirus titers were measured by using luciferase substrate (Promega). VSV-G pseudovirus was used as a negative control, whose envelope protein was different from the H5N1 pseudovirus. It was produced in the same manner as for the H5N1 pseudovirus.
For the pseudovirus neutralization assay, VSV-G or H5N1 pseudovirus was incubated with different concentrations of 1 for 30 min at 37 °C, followed by addition to MDCK cells for an additional 48 h. Subsequently, supernatants were discarded and the cell lysates were transferred to 96-well white plates. The luciferase substrate was added into the plates. The luciferase activity was measured by a Tecan Genios Pro microplate reader. HA Inhibition Assay. The inhibitory effect of 1 on the HA subunit, which mediates viral adsorption to the cellular sialic acid receptor, was evaluated by the HA inhibition assay as previously described.38,42 Briefly, 25 μL of 2-fold serial concentrations of 1 was mixed with the influenza virus PR8 at different titers for 30 min at room temperature. Subsequently, the mixture was incubated with 50 μL of 0.5% chicken erythrocytes at room temperature, and the results of HA inhibition were observed after incubation of the plate for 1 h at 37 °C. The viral diluent without 1 was used as the negative control, while PBS was used as the blank control. NA Inhibition Assay. The impact of 1 on viral particle release was assessed by the NA inhibition assay according to our previous description.43 Briefly, 15 μL of influenza virus A/Puerto Rico/8/3 4 (H1N1) solution diluted in the reaction buffer was mixed with 5 μL of 2-fold diluted 1 for 30 min at 37 °C in a 96-well black plate, and then 30 μL of 20 μM 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium (Sigma-Aldrich) dissolved in the reaction buffer was added. After incubation for 1 h at 37 °C, 50 μL of 14 mM NaOH was used to terminate the reaction. Finally, the fluorescence intensity of the product 4-methylumbelliferone was measured (340 nm as excitation wavelength and 440 nm as emission wavelength) using an Infinite M1000Pro. HTRF Assay. The HTRF assay was used to evaluate the effect of 1 on the PB2 protein activity as previously described.44 Briefly, the PB2 protein was mixed with 1 at different concentrations and incubated for 15 min at room temperature. After that, a premixed fluorescent probe solution was added, and the results were analyzed by a microplate reader after 1 h. SPR Assay. The binding affinity of 1 and PB2-cap protein was analyzed using a Biacore T100 system (GE Healthcare).40,45 Briefly, 0.5 μM PB2-cap protein was immobilized on a CM5 sensor chip (GE Healthcare) using a pH 5.0 acetic acid solution (Carlo Erba). Compound 1 at different concentrations (7.8, 15.6, 31.3, 62.5, and 125 nM) was injected for 180 s at a flow rate of 50 μL/min. A cleaning solution (50 mM NaOH) was used to regenerate the chip. The data were collected at 25 °C. KD values were calculated using Biacore Evaluation Software 2.0. The small molecule D715-2441 recently reported in the literature was utilized as the reference compound.37 Docking Method. The crystal structure of influenza A H5N1 PB2-cap binding domain with bound m7GTP was downloaded from RCSB Protein Data Bank (PDB code: 4CB4, resolution: 1.60 Å). UCSF Chimera was used to prepare the structures of protein and ligand. Hydrogens were added by using the Dock Prep module.46 The binding domain and ligand were assigned AMBER ff14SB force field and AM1-BCC charges, respectively.47,48 The box center was set as (49.994, −8.510, −3.792), and the sizes in the three directions were set as (20.737, 28.668, 24.377). The molecular surface of the binding domain was generated by the DMS tool in Chimera using a probe atom with a 1.4 Å radius, and then spheres filling the active site were produced by the Sphgen module. The Grid module was used to generate the Grid file, based on which Grid energy was evaluated. Semiflexible dockings were conducted with 10 000 different orientations using the DOCK 6.7 program.49 van der Waals and electrostatic interactions were obtained between the ligand and the binding site, summing up to Grid scores. Once finished, clustering analysis was performed (RMSD threshold was set at 2.0 Å) to obtain the best scored poses. Protein ligand interactions were analyzed by the online tool PLIP (https://projects.biotec.tu-dresden.de/plip-web/ plip/index) and visualized with PyMOL.50 Statistical Analysis. The statistical analysis of the data was carried out by Student’s t test with GraphPad Prism software. A p value of
