Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2–9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 μg/mL), which is better than that of the positive control, 2′CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS–NP computational analysis. The ChemGPS–NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space.


Introduction
Zoanthid of the genus Palythoa (family Sphenopidae) is a kind of benthos commonly found in shallow waters. More than 90 species of this genus were identified in subtropical and tropical areas all over the world. This marine creature is characterized by absorbing sands or small sediments into polyp to reinforce their structure. Apart from the well-known poisonous compound, palytoxin [1], Palythoa zoanthids were also reported to produce various natural products, such as amino acids [2,3], steroids [4], ecdysteroids [5], prostanoids [6], and sulfonylated ceramides [7]. The natural products of zoanthids not only act as defensive substance against predators, but also exhibit diverse bioactivities for the development of new drugs. For example, a polyhydroxylated steroid isolated from P. tuberculosa selectively inhibited human breast cancer cells (MCF-7), which implied this compound might be a new anti-cancer therapeutic agent [8]. Recently, our research group has studied anti-dengue virus ecdysteroids from Formosan zoanthid Zoanthus spp. [9]. In our continuous screening for bioactive marine natural products, the ethanolic extract of P. mutuki showed strong anti-dengue virus activity. Because there is no medicine for dengue fever, the animal materials of P. mutuki were investigated for its bioactive ingredients. In this manuscript, the isolation, structural elucidation, antiviral activity, and ChemGPS-NP space mapping analysis of one new and eight known compounds from P. mutuki are described.

Introduction
Zoanthid of the genus Palythoa (family Sphenopidae) is a kind of benthos commonly found in shallow waters. More than 90 species of this genus were identified in subtropical and tropical areas all over the world. This marine creature is characterized by absorbing sands or small sediments into polyp to reinforce their structure. Apart from the well-known poisonous compound, palytoxin [1], Palythoa zoanthids were also reported to produce various natural products, such as amino acids [2,3], steroids [4], ecdysteroids [5], prostanoids [6], and sulfonylated ceramides [7]. The natural products of zoanthids not only act as defensive substance against predators, but also exhibit diverse bioactivities for the development of new drugs. For example, a polyhydroxylated steroid isolated from P. tuberculosa selectively inhibited human breast cancer cells (MCF-7), which implied this compound might be a new anti-cancer therapeutic agent [8]. Recently, our research group has studied anti-dengue virus ecdysteroids from Formosan zoanthid Zoanthus spp. [9]. In our continuous screening for bioactive marine natural products, the ethanolic extract of P. mutuki showed strong anti-dengue virus activity. Because there is no medicine for dengue fever, the animal materials of P. mutuki were investigated for its bioactive ingredients. In this manuscript, the isolation, structural elucidation, antiviral activity, and ChemGPS-NP space mapping analysis of one new and eight known compounds from P. mutuki are described.
Due to the anti-dengue virus activities of some ecdysteroids (5-8) that were revealed previously [9], the other five compounds (1-4, and 9) were selected to evaluate their anti-dengue virus 2 (DENV-2) activities and cytotoxicity. As a result, palythone A (1) and 24-epi-makisterone (4) demonstrated weak anti-DENV-2 activities (Table 2). Unexpectedly, compound 9 exhibited the most potent antiviral activity with an EC50 value of 4.50 ± 0.46 μM. This activity is superior to that of the positive control, 2′CMC, which was previously reported as a specific anti-DENV agent in vitro and in vivo [18].   [17]. Therefore, structure of 1 was determined unambiguously.
Due to the anti-dengue virus activities of some ecdysteroids (5-8) that were revealed previously [9], the other five compounds (1-4, and 9) were selected to evaluate their anti-dengue virus 2 (DENV-2) activities and cytotoxicity. As a result, palythone A (1) and 24-epi-makisterone (4) demonstrated weak anti-DENV-2 activities (Table 2). Unexpectedly, compound 9 exhibited the most potent antiviral activity with an EC 50 value of 4.50˘0.46 µM. This activity is superior to that of the positive control, 2 1 CMC, which was previously reported as a specific anti-DENV agent in vitro and in vivo [18].   [17]. Therefore, structure of 1 was determined unambiguously.
Due to the anti-dengue virus activities of some ecdysteroids (5-8) that were revealed previously [9], the other five compounds (1-4, and 9) were selected to evaluate their anti-dengue virus 2 (DENV-2) activities and cytotoxicity. As a result, palythone A (1) and 24-epi-makisterone (4) demonstrated weak anti-DENV-2 activities (Table 2). Unexpectedly, compound 9 exhibited the most potent antiviral activity with an EC50 value of 4.50 ± 0.46 μM. This activity is superior to that of the positive control, 2′CMC, which was previously reported as a specific anti-DENV agent in vitro and in vivo [18].    In addition, the ability to suppress virus production of peridinin (9) was measured by a TCID50 assay. The result showed that 9 at dose of 10 μM effectively decreased the viral titer by 2 ± 0.6 log10 as compared to mock-treatment. Peridinin (9) was also tested for other sero-types of DENV, and the results are shown in Table 3. The results indicated that it can inhibit all sero-types of DENV. Moreover, the anti-DENV protease activity of peridinin (9) was characterized by using NS3 protease reporter-based assay. The results showed this compound exhibited inhibitory effect on DENV protease activity with an EC50 value of 8.50 ± 0.41 μM. To date, no approved agents for treating DENV infection is available, thus there is an urgent need to develop potential anti-DENV agents. Currently, some natural products have been identified to exhibit anti-DENV activity. For example, Zandi et al. have reported that the Scutellaria baicalensis extract and quercetin exhibited anti-DENV activity with IC50 value of 93.66 μg/mL (SI = 9.74) and 35.7 μg/mL (SI = 7.07), respectively [19,20]. In addition, Brandão et al. have identified the anti-DENV activity of Arrabidaea pulchra extract with an IC50 value of 46.8 ± 1.6 μg/mL (SI = 2.7) [21]. Those natural products exhibited lower anti-DENV activity and SI value than that of peridinin (9). To determine the applicability of peridinin (9), the in vivo tests will be performed in the future. Furthermore, future studies about  In addition, the ability to suppress virus production of peridinin (9) was measured by a TCID 50 assay. The result showed that 9 at dose of 10 µM effectively decreased the viral titer by 2˘0.6 log 10 as compared to mock-treatment. Peridinin (9) was also tested for other sero-types of DENV, and the results are shown in Table 3. The results indicated that it can inhibit all sero-types of DENV. Table 3. Anti-DENV-1-4 activity of peridinin (9). Moreover, the anti-DENV protease activity of peridinin (9) was characterized by using NS3 protease reporter-based assay. The results showed this compound exhibited inhibitory effect on DENV protease activity with an EC 50 value of 8.50˘0.41 µM. To date, no approved agents for treating DENV infection is available, thus there is an urgent need to develop potential anti-DENV agents. Currently, some natural products have been identified to exhibit anti-DENV activity. For example, Zandi et al. have reported that the Scutellaria baicalensis extract and quercetin exhibited anti-DENV activity with IC 50 value of 93.66 µg/mL (SI = 9.74) and 35.7 µg/mL (SI = 7.07), respectively [19,20]. In addition, Brandão et al. have identified the anti-DENV activity of Arrabidaea pulchra extract with an IC 50 value of 46.8˘1.6 µg/mL (SI = 2.7) [21]. Those natural products exhibited lower anti-DENV activity and SI value than that of peridinin (9). To determine the applicability of peridinin (9), the in vivo tests Mar. Drugs 2016, 14, 151 6 of 10 will be performed in the future. Furthermore, future studies about computer-aided development of pharmacophoric models should be performed to optimize the antiviral activity of peridinin (9). Since there is no antiviral drug against DENV infection, our study identifies a potential lead compound for novel anti-DENV agent development.
On the basis of the chemical space concept, a computational high-throughput screening method named the ChemGPS-NP was advanced by Josefin et al. [22,23]. ChemGPS-NP is a principal component analysis (PCA) based coordinate system with eight dimensions, which mainly concerns the size, shape, and polarizability (PC1), aromatic-and conjugation-related properties (PC2), lipophilicity, polarity, and H-bond capacity (PC3), and flexibility (PC4) of natural products. There is a theory which states that compounds with similar physico-chemical properties could possess comparable bioactivities and mechanisms [24]. Therefore, the molecular diversity of the isolates and three anti-dengue virus datasets was analyzed by the ChemGPS-NP map of chemical space. The first dataset consisted of the seventeen anti-dengue virus ecdysteroids isolated from zoanthids; the second dataset contained eight anti-dengue virus limonoids separated from Swietenia macrophylla [25]; and the third dataset composed of twelve non-peptidic anti-dengue virus compounds [26,27]. The score plot of three descriptors (PC1, PC2, and PC4) revealing that peridinin (9), limonoids, and ecdysteroids situated in the same quadrant ( Figure 5), and, meanwhile, peridinin and limonoids occupied the same quadrant while the descriptors changed to PC1, PC2, and PC3. Furthermore, two positive control compounds (ribavirin and 2 1 CMC) and twelve non-peptidic anti-dengue virus compounds placed in different quadrants away from all natural products. Our findings suggested natural products located in the specific chemical space might be possible new anti-dengue virus agents.
Mar. Drugs 2016, 14, 131 6 of 10 computer-aided development of pharmacophoric models should be performed to optimize the antiviral activity of peridinin (9). Since there is no antiviral drug against DENV infection, our study identifies a potential lead compound for novel anti-DENV agent development.
On the basis of the chemical space concept, a computational high-throughput screening method named the ChemGPS-NP was advanced by Josefin et al. [22,23]. ChemGPS-NP is a principal component analysis (PCA) based coordinate system with eight dimensions, which mainly concerns the size, shape, and polarizability (PC1), aromatic-and conjugation-related properties (PC2), lipophilicity, polarity, and H-bond capacity (PC3), and flexibility (PC4) of natural products. There is a theory which states that compounds with similar physico-chemical properties could possess comparable bioactivities and mechanisms [24]. Therefore, the molecular diversity of the isolates and three anti-dengue virus datasets was analyzed by the ChemGPS-NP map of chemical space. The first dataset consisted of the seventeen anti-dengue virus ecdysteroids isolated from zoanthids; the second dataset contained eight anti-dengue virus limonoids separated from Swietenia macrophylla [25]; and the third dataset composed of twelve non-peptidic anti-dengue virus compounds [26,27]. The score plot of three descriptors (PC1, PC2, and PC4) revealing that peridinin (9), limonoids, and ecdysteroids situated in the same quadrant ( Figure 5), and, meanwhile, peridinin and limonoids occupied the same quadrant while the descriptors changed to PC1, PC2, and PC3. Furthermore, two positive control compounds (ribavirin and 2′CMC) and twelve non-peptidic anti-dengue virus compounds placed in different quadrants away from all natural products. Our findings suggested natural products located in the specific chemical space might be possible new anti-dengue virus agents.

General Experimental Procedures
Optical rotation was measured on a JASCO P-1020 digital polarimeter (Tokyo, Japan). UV data were recorded on a JASCO V-530 UV/VIS Spectrophotometer (Tokyo, Japan). CD spectrum was acquired on a JASCO J-815 CD spectrometer (Tokyo, Japan). High-resolution ESIMS data were obtained on a Bruker APEX II spectrometer (Billerica, MA, USA). IR spectrum was measured on a Perkin Elmer system 2000 FT-IR spectrophotometer (Waltham, MA, USA). NMR spectra were obtained by Varian 600 MHz NMR (San Carlos, CA, USA). Merck silica gel 60 (Billerica, MA, USA) and Sephadex LH-20 (Stockholm, Sweden) were used for column chromatography. The instrumentation for HPLC was composed of a Shimadzu LC-10AD pump (Kyoto, Japan) and a Shimadzu SPD-M10A PDA detector (Kyoto, Japan).

Animal Material
Specimens of Palythoa mutuki were collected in Keelung City, Taiwan, in August 2015. The research samples were identified by its 16S rDNA gene sequence. A voucher specimen (no. KMU-MrPm) was deposited in the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University.

ECD Calculations
The minima energies of 4R-1 and 4S-1 were calculated by ChemBio3D (ver. 14.0, Perkin Elmer, Waltham, MA, USA) and the structure of 4R-1 and 4S-1 were saved as tinker MM2 input files. These data were imported into the Gaussian 09 for density functional theory (DFT) at the B3LYP/6-31G(d) level in the gas phase to obtain the restricted conformations. The energies, rotational strengths, and oscillator strengths of the 20 weakest conformers were optimized using the time-dependent density functional theory (TDDFT) methodology at the B3LYP/6-311++G(d,p) level. The final ECD files were converted to txt files by GaussSum 2.2.5 (Gaussian Inc., Wallingford, CT, USA) with a bandwidth σ of 0.5 eV. The ECD and CD curves were plotted by Excel.

Anti-DENV Activity Assay
Huh-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% antibiotic-antimycotic in a 5% CO 2 at 37˝C. Huh-7 cells were seeded at 24-well plate at density 5ˆ10 4 cells/well and infected by DENV infection at a multiplicity of infection (MOI) of 0.2 followed by test compounds treatment 2 h post infection. Total cellular RNA were harvested at 72 h post-infection, and DENV RNA level was analyzed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) with specific primers against DENV NS5 gene. DENV RNA level was normalized by cellular glyceraldehydes-3-phosphate dehydrogenase (gapdh) mRNA level.

Evaluation of Anti-DENV RdRp and Protease Activity
The DENV RdRp activity reporter system was used to determine the anti-DENV RdRp activity as described before [18]. The NS3 protease reporter-based assay was used to determine the anti-DENV protease activity. The Huh-7 cells were transfected with DENV NS2B/NS3 protease reporter vector pEG(∆4B/5)NLuc carrying a specific DENV protease cleavage peptide sequences and the DENV protease expression vector. Subsequently, the cells were treated with compound 9 for 3 days. The supernatant was collected to analyze the nano luciferase (NLuc) activity by Nano-Glo ® Luciferase Assay System following the manufacturer's instructions (Promega, Madison, WI, USA).

Cytotoxicity Assay
Huh-7 cells were seeded onto 96-well plate at a density of 5ˆ10 3 cells per well, followed by compound treatment for 72 h. The cell viability was determined using a standard MTS assay (CellTiter 96 ® Aqueous One Solution Cell Proliferation assay system, Promega, Madison, WI, USA) according to the manufacturer's instructions.

Conclusions
In our continuous investigation on discovering anti-dengue virus natural products, the Formosan zoanthid Palythoa mutuki has resulted in the identification of one new ecdysteroid (1) and eight known compounds (2)(3)(4)(5)(6)(7)(8)(9). The potent anti-dengue virus activity of peridinin (9), a common secondary metabolite in marine invertebrates and dinoflagellates was discovered for the first time. Our findings suggest carotenoid-like substance might possess anti-dengue virus activity. Zoanthids have become one of the good resources for antiviral natural product development.