Napyradiomycin A4 and Its Relate Compounds, a New Anti-PRV Agent and Their Antibacterial Activities, from Streptomyces kebangsaanensis WS-68302

Two new napyradiomycins derivatives, napyradiomycin A4 (1) and A80915 H (2), along with five known ones, were isolated from the ethyl acetate extract of fermentation culture of Streptomyces kebangsaanensis WS-68302. Their structures were elucidated by extensive spectroscopic analysis, including HR-MS, 1D and 2D NMR, CD spectrum, as well as comparison with literature data. Compound 1 exhibited significant antiviral activity against PRV (Pseudorabies virus) with an IC50 value of 2.056 μM and therapeutic ratio at 14.98, suggesting that it might have potential for development of an antiviral agent. Moreover, compound 1 displayed the strongest inhibition against PRV protein among the tested napyradiomycins in the indirect immunofuorescence assay. Compounds 3 and 4 showed higher activities against swine pathogenic Streptococcus suis than the positive control penicillin G sodium salt, with MIC values of 3.125 and 6.25 μg/mL, respectively. Compounds 1 and 3–6 exhibited moderate antibacterial activity against the swine pathogenic Erysipelothrix rhusiopathiae, with MIC values ranging from 25 to 50 μg/mL.


Introduction
In recent decades, infection with pathogens has caused huge economic losses to the swine industry. Pseudorabies virus (PRV), a herpes virus, is one of the most important pathogens in swine [1,2]. The swine pseudorabies caused by the pseudorabies virus is characterized by reproductive disturbance, dyspnea, and neurological symptoms, and different-aged pigs are susceptible to infection with the disease [3,4]. Vaccination is one of the most important methods to prevent and control PRV; however, with the emergence of PRV mutant strains in recent years, it is difficult to completely prevent and control them using conventional vaccines [5][6][7][8]. Moreover, no effective anti-PRV drugs have been approved [9]. Therefore, it is essential to discover antiviral drugs.
In addition to the virus, some bacteria, such as Streptococcus suis and Erysipelothrix rhusiopathiae, can also cause economic losses and zoonotic diseases. S. suis is an important zoonotic pathogen that causes several pathological conditions such as septicemia, meningitis, arthritis and pneumonia [10]. S. suis can cause bacterial encephalitis or toxic-shock-like syndrome in humans [11]. E. rhusiopathiae, a small Gram-positive bacterium, is also a zoonotic pathogen [12]. The swine erysipelas caused by E. rhusiopathiae mainly has acute, subacute and chronic clinical forms, and acute infection can even cause sudden death or general signs of septicemia to swine [13]. E. rhusiopathiae can also cause a skin disease, erysipeloid, in humans [13]. Although many antibiotic drugs can treat bacterial diseases, with the emergence bacterial resistance, the development of new drugs to prevent and treat these diseases is urgent [14]. disease, erysipeloid, in humans [13]. Although many antibiotic drugs can treat bacteri diseases, with the emergence bacterial resistance, the development of new drugs to pr vent and treat these diseases is urgent [14].
In our ongoing research on the bioactive secondary metabolites from actinomycete a soil-derived strain WS-68302 was found to show interesting antiviral activity again PRV and antibacterial activity. The actinomycete strain was identified as S. keban saanensis WS-68302. Further chemical investigation of the extract from the strain led the discovery of two new derivatives of napyradiomycins, named napyradiomycin A4 ( and A80915H (2), as well as five known ones (3-7) ( Figure 1). The napyradiomycins and 3-6) showed strong anti-PRV activity in vitro. In this study, we report the taxonom of the strain, the isolation, the structural elucidation, and the antiviral and antibacteri activities of these compounds from the strain.

Taxonomy of Strain WS-68302
The colonies of strain WS-68302 on ISP-2 agar grew abundant grayish white myc lium (Figure 2A). The aerial mycelia were straight to flexuous and spiral on terminal ( Figure 2B). The spores were mostly ovoid and 0.8-1.2 × 1.2-2.0 μm in size, and the su

Taxonomy of Strain WS-68302
The colonies of strain WS-68302 on ISP-2 agar grew abundant grayish white mycelium ( Figure 2A). The aerial mycelia were straight to flexuous and spiral on terminals. ( Figure 2B). The spores were mostly ovoid and 0.8-1.2 × 1.2-2.0 µm in size, and the surfaces of the spores were covered with different-length spines ( Figure 2C). These morphological characteristics of strain WS-68302 were in total agreement with the strain Streptomyces kebangsaanensis SUK12, which was identified as a novel species of the genus Streptomyces in 2013, isolated from Malaysian ethnomedicinal plant Portulaca olerace [27]. The 16S rRNA sequence of the strain WS-68302 was deposited in the NCBI GenBank database (accession number OP787989), and the BLAST result of the sequence also verified the closest similarity (98.99%) to that of S. kebangsaanensis SUK12. The phylogenetic tree generated using the neighborjoining method clearly depicted the evolutionary relationship of the strain WS-68302 with Streptomyces ( Figure 3). Accordingly, the strain WS-68302 was identified as S. kebangsaanensis and designated S. kebangsaanensis WS-68302.

PEER REVIEW
3 of 12 faces of the spores were covered with different-length spines ( Figure 2C). These morphological characteristics of strain WS-68302 were in total agreement with the strain Streptomyces kebangsaanensis SUK12, which was identified as a novel species of the genus Streptomyces in 2013, isolated from Malaysian ethnomedicinal plant Portulaca olerace [27]. The 16S rRNA sequence of the strain WS-68302 was deposited in the NCBI GenBank database (accession number OP787989), and the BLAST result of the sequence also verified the closest similarity (98.99%) to that of S. kebangsaanensis SUK12. The phylogenetic tree generated using the neighbor-joining method clearly depicted the evolutionary relationship of the strain WS-68302 with Streptomyces ( Figure 3). Accordingly, the strain WS-68302 was identified as S. kebangsaanensis and designated S. kebangsaanensis WS-68302.

Structural Elucidation
Compound 1 was obtained as pale yellow oil. In the electrospray ionization mass spectrometry spectrum (ESIMS), compound 1 showed a positive ion cluster at m/z  Molecules 2023, 28, x FOR PEER REVIEW faces of the spores were covered with different-length spines (Figur phological characteristics of strain WS-68302 were in total agreeme Streptomyces kebangsaanensis SUK12, which was identified as a novel sp Streptomyces in 2013, isolated from Malaysian ethnomedicinal plant Po The 16S rRNA sequence of the strain WS-68302 was deposited in the N tabase (accession number OP787989), and the BLAST result of the sequ the closest similarity (98.99%) to that of S. kebangsaanensis SUK12. The generated using the neighbor-joining method clearly depicted the tionship of the strain WS-68302 with Streptomyces ( Figure 3). Accor WS-68302 was identified as S. kebangsaanensis and designated WS-68302.
The absolute configurations at C-2 and C-3 of 2 were determined to be 2S and 3S by comparing its CD spectrum ( Figure S25) with that of A80915G ( Figure S26), whose structure was confirmed by spectroscopic data and chemical synthesis [19,28]. However, the absolute configuration of C-4" was not determined due to scarcity of 2 for modified Mosher's analysis [29].
Compounds 3-7 were identified as napyradiomycin A1 (3) [16], napyradiomycin B1 (4) [16], A80915G (5), A80915G-8"-acid (6) [30], and naphthomevalin (7) [31,32] (Figure 1), respectively, by comparing their MS, 1 H and 13 C MMR spectroscopic data with those previously reported. The in vitro antiviral activities of 1-7 against PRV in Marc-145 cells were evaluated by MTT assay according to the protocol. Compounds 1 and 3-6 exhibited significant antiviral activity against PRV, with half maximal inhibitory concentration (IC 50 ) values ranging from 2.056 to 26.47 µM, whereas 2 and 7 did not display any apparent anti-PRV activity ( Table 3). It is notable that compounds 1 and 3 exhibited excellent antiviral activity against PRV, with IC 50 values of 2.056 and 2.208 µM, respectively-much stronger than that of the positive control ribavirin (IC 50 = 58.032 µM). However, the new compound 1 showed a higher therapeutic ratio (TI = 14.98) than 3 (TI = 2.62). Although their structures are closely similar, there is a carbonyl group at C-16 for compound 1 and a double bond at C-16 and C-17 for compound 2, which led to a difference in toxicity between the two compounds and indicated that the substituent of the 16 position is important for toxicity. Despite compound 5 demonstrating a strong activity against PRV with IC 50 value of 8.26 µM, its new derivative 2 was inactive against PRV, which suggested the hydroxyl substitution at C-4" and C-8" of 5 had an effect on the anti-PRV activity. Compounds 4 and 6 displayed a lower therapeutic index (TI < 2.0). As a result, compound 1 might have potential for the development of an antiviral agent.

Indirect Immunofluorescence Assay
Based on the above antiviral activity, we selected the active compounds 1, 3 and 4 to test the immunofluorescence effect at concentrations of 2, 5 and 10 µg/mL, respectively, by indirect immunofluorescence assay. The three compounds were co-incubated with PRV in Marc-145 cells, respectively. After 24 h, the nucleocapsid (DAPI, blue) and PRV (FITC, green) responses to the indirect immunofluorescence effect are shown in Figure 5. The green fluorescence intensity in the compound-treated cells ( Figure 5B-D) was obvious lower than in the virus control group ( Figure 5A), indicating that the viral protein synthesis was significantly suppressed by the three active compounds, 1, 3 and 4. Furthermore, the new compound 1 showed lower immunofluorescence effect against PRV and lower cytotoxicity than the known compounds 3 and 4 in the Marc-145 cells.

Antibacterial Activities
Compounds 1-7 were evaluated using a microdilution broth method for antibacterial activity against S. aureus and the animal pathogens S. suis, E. rhusiopathiae, and E. coli. Penicillin G sodium salt and cefpirome sulfate were used as positive controls. As shown in Table 4, compounds 3, 4 and 7 displayed potent activities comparable to the positive control cefpirome sulfate (MIC < 0.78 μg/mL) against bacteria S. aureus ATCC 25923, while the new compounds 1 and 6 showed moderate activity against the bacteria, with MIC values of 25 and 50 μg/mL, respectively. Compounds 3 and 4 also showed higher activities against the swine pathogenic S. suis than the positive control penicillin G sodium salt, with MIC values of 3.125 and 6.25 μg/mL, respectively, whereas the new compound 1 exhibited weak activity against the bacteria. Compounds 1 and 3-6 exhibited moderate antibacterial activity against the swine pathogenic E. rhusiopathiae, with MIC values ranging from 25 to 50 μg/mL. There are slight differences on a side chain in the structures of compounds 2, 5 and 6, but compound 2 did not show any activity against the three tested Gram-positive bacteria. Compound 5, previously isolated from Streptomyces aculeolatus A80915, had good antibacterial activity against aerobic Gram-positive Staphylococcus, Streptococcus and Enterococcus bacterial strains (MIC 2-16 μg/mL) [19]. While compound 6, an oxidized compound of 5, only had weak antibacterial activity against Bacillus subtilis ATCC6633 (MIC 64 μg/mL) [30]. Therefore, it was suggested that hydroxyl substitution at C-4′′ and C-8′′of the side chains on the semi-naphthoquinone nucleus had a great influence on the activities of compound 2. As

Antibacterial Activities
Compounds 1-7 were evaluated using a microdilution broth method for antibacterial activity against S. aureus and the animal pathogens S. suis, E. rhusiopathiae, and E. coli. Penicillin G sodium salt and cefpirome sulfate were used as positive controls. As shown in Table 4, compounds 3, 4 and 7 displayed potent activities comparable to the positive control cefpirome sulfate (MIC < 0.78 µg/mL) against bacteria S. aureus ATCC 25923, while the new compounds 1 and 6 showed moderate activity against the bacteria, with MIC values of 25 and 50 µg/mL, respectively. Compounds 3 and 4 also showed higher activities against the swine pathogenic S. suis than the positive control penicillin G sodium salt, with MIC values of 3.125 and 6.25 µg/mL, respectively, whereas the new compound 1 exhibited weak activity against the bacteria. Compounds 1 and 3-6 exhibited moderate antibacterial activity against the swine pathogenic E. rhusiopathiae, with MIC values ranging from 25 to 50 µg/mL. There are slight differences on a side chain in the structures of compounds 2, 5 and 6, but compound 2 did not show any activity against the three tested Gram-positive bacteria. Compound 5, previously isolated from Streptomyces aculeolatus A80915, had good antibacterial activity against aerobic Gram-positive Staphylococcus, Streptococcus and Enterococcus bacterial strains (MIC 2-16 µg/mL) [19]. While compound 6, an oxidized compound of 5, only had weak antibacterial activity against Bacillus subtilis ATCC6633 (MIC 64 µg/mL) [30]. Therefore, it was suggested that hydroxyl substitution at C-4" and C-8"of the side chains on the semi-naphthoquinone nucleus had a great influence on the activities of compound 2. As reported in the literature [15,17,19], these tested compounds were inactive to the Gram-negative bacteria E. coli ATCC 25922.

Conclusions
In conclusion, seven napyradiomycins, including two new ones, napyradiomycin A4 (1) and A80915H (2), were obtained from the fermentation culture of Streptomyces kebangsaanensis WS-68302. To the best of our knowledge, this is the first time that the potent antiviral activities of napyradiomycins against PRV in vitro have been observed. Impressively, the new compound, napyradiomycin A4 (1), and napyradiomycin A1 (3) both showed much better inhibitory activity against PRV than ribavirin. In addition, napyradiomycin A1 (3) and B1 (4) and naphthomevalin (7) displayed potent activities comparable to the positive control cefpirome sulfate against the bacteria S. aureus. Compounds 3 and 4 also showed higher activities against swine pathogen S. suis than the positive control penicillin G sodium salt. This study suggested that the napyradiomycin A4 could serve as a promising antiviral agent against PRV.

Isolation of Strain and Morphological Identification
The strain WS-68302 was isolated from a soil sample collected from a hillside in SuiXian County, Hubei Province, China, in April 2015. For the taxonomic studies of the actinomycetes, ISP-2 agar (glucose 0.4%, malt extract 1%, yeast extract 0.4%, agar 1.8%, and pH 7.2) was used to investigate the morphological and physiological characteristics. The strain was incubated at 28°C for 2-3 weeks. Morphological properties were observed under a scanning electron microscope (Model Hitachi SU8100). Genomic DNA extraction, PCR amplification and sequencing of 16S rRNA were developed with the universal methods [33]. The strain was identified based on sequence analysis of 16S rRNA Sequence under the BLAST search.

Fermentation
A stock culture of the strain S. kebangsaanensis WS-68302 was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of the seed medium ISP-2. The flasks were incubated at 28°C on a rotary shaker at 150 rpm, for 96 h. 10% seed culture was transferred to 500 mL Erlenmeyer flasks containing 100 mL of the production medium ISP-2 under sterile conditions. The flasks were cultivated at 28°C on a rotary shaker at 150 rpm for 120 h.

Cell Proliferation
The supernatant was discarded upon Marc proliferation; cells were washed with PBS, and trypsin was added until the cells were digested into singles at 37°C. Cells were dispersed in DMEM with 10% FBS and cultured at 37°C for 2-3 days.

Cell Bioability Assay
Cell viability was assessed using the MTT according to the protocol. Briefly, cells in 96-well plates were treated with compounds in different concentration for 48 h. Assays were performed in triplicate. Then, MTT was added to the cells and they were incubated in the dark for 4 h at 37°C. The supernatant was discarded and DMSO was added. After 10 min incubation, the OD 450 of each well was read using a spectrophotometer (Thermo). The cell viability (%) was calculated as (compound average OD 450µM value/cell control average OD 450µM value) × 100%. Compounds were added to monolayer in different concentration. After 48h incubation, the cell viability (%) was calculated by MTT assay, and the cytotoxic effect (CC 50 ) was analyzed by SPSS 16.0 software. Compounds in different concentrations (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 µg/mL) and PRV (100 TCID 50 ) were added to monolayer at 37°C. After 72h incubation, the cell viability (%) was calculated by MTT assay, and the half maximal inhibitory concentration (IC 50 ) was analyzed by SPSS 16.0 software. Ribavirin was used as a positive control.

Indirect Immunofluorescence Assay
The monolayers of Marc-145 cells cultured in 24-well plates were treated with PRV and drug mixture. After 24 h incubation, the cells were washed with PBS twice and fixed with cold absolute ethanol at 4 • C for 15 min, then permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h. The cells were then incubated with 1:200 anti-Pseudorabies Virus antibody in PBS containing 1% BSA at 4 • C overnight. After washing three times with PBST, the cells were stained with 1:2000 goat anti-rabbit IgG H&L. To show the nucleus of the cells, DAPI (40, 6-diamidino-2-phenylindole) was used to stain the cellular DNA. The fluorescent images were observed using an inverted fluorescence microscope (Olympus IX73 (Olympus Corporation, Tokyo, Japan)).

Antibacterial Assays
The minimum inhibitory concentrations of 1-7 were determined against the four bacterial strains (Staphylococcus aureus ATCC 25923, Erysipelothrix rhusiopathiae WH13013, Streptococcus suis SC19, Escherichia coli ATCC 25922) following the Clinical and Laboratory Standards Institute (CLSI) guidelines [34]. The microdilution broth method was performed in 96-well plates (Corning Costar ® 3599 Corning, Corning, NY, USA) using MHB (Hopebio, Qingdao, China). The final concentration of the culture was 5 × 10 5 colony-forming units (CFU)/mL, and measurements were repeated at least in triplicate. Penicillin G sodium salt and cefpirome sulfate were purchased from Solarbio.