Anti-Human Rhinoviral Activity of Polybromocatechol Compounds Isolated from the Rhodophyta, Neorhodomela aculeata

An extract of the red alga, Neorhodomela aculeata, exhibited antiviral activity against human rhinoviruses. Bioassay-guided purification was performed to yield six compounds, which were subsequently identified as lanosol (1) and five polybromocatechols (2–6) by spectroscopic methods, including 1D and 2D NMR and mass spectrometric analyses. Structurally, all of these compounds, except compound 5, contain one or two 2,3-dibromo-4,5-dihydroxyphenyl moieties. In a biological activity assay, compound 1 was found to possess antiviral activity with a 50% inhibitory concentration (IC50) of 2.50 μg/mL against HRV2. Compound 3 showed anti-HRV2 activity, with an IC50 of 7.11 μg/mL, and anti-HRV3 activity, with an IC50 of 4.69 μg/mL, without demonstrable cytotoxicity at a concentration of 20 μg/mL. Collectively, the results suggest that compounds 1 and 3 are candidates for novel therapeutics against two different groups of human rhinovirus.


Introduction
More than 500 species of marine algae are distributed along the coast of Korea. Some of these algae are sources of foods or traditional medicines [1], while others may serve as important resources for bioactive natural products [2][3][4]. Red algae of the Rhodomelaceae (Ceramiales) family are rich sources of several monoaryl, diaryl, and triaryl bromocatechol structural types with various biological activities, including anticancer, antioxidative, antimicrobial, and anti-thrombotic effects [5][6][7][8][9]. However, there have been no reports describing the characterization of compounds derived from Neorhodomela aculeata (L.P. Perestenko) Masuda. (Rhodomelaceae) collected off the Korean coast. A few reports have described the antioxidative and antibacterial activities of methanolic extracts of N. aculeata, but there have not been reports of any antiviral activity associated with these extracts [10].
Human rhinoviruses (HRVs), members of the Picornaviridae family, are divided into three distinct species: type A, type B, and type C. These viruses are the predominant causal agents of viral respiratory tract infections, particularly common colds [11], as well as acute otitis media and sinusitis [12,13]. Specific and small-molecule antiviral agents for the treatment of picornavirus infections are not currently available. The production of vaccines to prevent rhinovirus infections is also known to be very challenging due to the more than 100 immunologically non-cross-reactive rhinovirus serotypes [14]. Therefore, most efforts have been focused on the development of effective antiviral agents for treating rhinovirus infections [15]. Several antiviral compounds have been shown to inhibit members of the picornavirus family by binding to the viral capsid proteins [16]. One of these, pleconaril, is a new-generation antiviral agent that has shown activity against rhinoviruses, excluding the 25 serotypes of HRV-B [17].
Herein, we report the structurals elucidation of polybromocatechol compounds isolated from N. aculeata and their anti-human rhinovirus activity.

Antiviral Activity and Cytotoxicity of Compound 1 and Compound 3 against HRV2 and HRV3
The antiviral activity of the six isolated compounds (1-6) was tested; compounds 2, 4, 5, and 6 showed no antiviral effect (data not shown). However, the antiviral assays demonstrated that compound 1 showed anti-HRV2 activity with a 50% inhibitory concentration (IC 50 ) value of 2.50 μg/mL and a 50% cytotoxic concentration (CC 50 ) value of more than 20 μg/mL, although it did not show anti-HRV3 activity (Table 4). Compound 3 also possessed strong antiviral activity with IC 50 values of 7.11 μg/mL against HRV2 and 4.69 μg/mL against HRV3, and a CC 50 value of more than 20 μg/mL (Table 4). Ribavirin, tested as a positive control, also showed antiviral activity in HeLa cells infected with HRV2 and HRV3 with IC 50 values of 2.15 μg/mL and 5.09 μg/mL, respectively, and exhibited a CC 50 value of more than 20 μg/mL (Table 4). After

Viruses, Cells and Reagents
HRV 2 and 3 were provided by the ATCC (American Type Culture Collection, Manassas, VA, USA) and were propagated in human epitheloid carcinoma cervix (HeLa) cells at 32 °C. HeLa cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 0.01% antibiotic-antimycotic.
Antibiotic-antimycotic, FBS and MEM were supplied by Gibco BRL (Grand Island, NY, USA). The tissue culture plates were purchased from Falcon (BD Biosciences, NJ, USA).

Assays of Antiviral Activity and Cytotoxicity
Assays of antiviral activity and cytotoxicity were evaluated by the SRB method using CPE reduction, recently reported [17]. Briefly, One day before infection, HeLa cells were seeded onto a 96-well culture plate at a concentration of 2 × 10 4 cells per well. Next day, medium was removed and then washed with 1× phosphate buffered saline (PBS). Infectivity of virus stock was determined by the SRB method using cytopathic effect (CPE) reduction and was determined as infectivity of the virus by SRB ID 50 (50% infective dose). Following this, 0.09 mL of diluted virus suspension of HRV2 or HRV3 containing CCID 50 (50% cell culture infective dose) of the virus stock to produce a appropriate cytopathic effects within 2 days after infection and 0.01 mL of medium supplemented with 20 mM MgCl 2 containing an appropriate concentration of the compounds were added. The antiviral activity of each test material was determined with a 10-fold diluted concentration ranging from 0.1 to 100 μg/mL. Three wells were used as virus controls (virus-infected non-drug-treated cells) while three wells were used as cell controls (non-infected non-drug-treated cells). The culture plates were incubated at 37 °C in 5% CO 2 for 2 days. After washing 1 times with 1× PBS, 100 μL of cold (−20 °C ) 70% acetone were added to each well and left for 30 min at −20 °C . 70% acetone was removed and 96-well plates were left at dry oven for 30 min. 100 μL of 0.4% (w/v) SRB in 1% acetic acid solution were added to each well and left at room temperature for 30 min. Unbound SRB was removed and the plates were washed 5 times with 1% acetic acid before oven drying and were then left in a dry oven for 1 day. Bound SRB was solubilized with 100 μL of 10 mM unbuffered tris-base solution and plates were left on a table for 30 min. The absorbance was read at 540 nm by using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance at 620 nm. To calculate the IC 50 values, the results were transformed to percentage of controls and the IC 50 values were graphically obtained from the dose-response curves. The percent protection achieved by the test compound in HRV2 or HRV3-infected cells was calculated by the following formula: where (OD t ) virus is the optical density measured with a given concentration of the test compound in virus-infected cells; (OD c ) virus is the optical density measured for the control untreated virus-infected cells; and (OD c ) mock is the optical density measured for control untreated mock-infected cells. The concentration achieving 50% protection according to the formula above was defined as the 50% inhibitory concentration (IC 50 ). The therapeutic index was defined as CC 50 /IC 50 . To measure cytotoxicity, HeLa cells were seeded onto a 96-well culture plate at a concentration of 2 × 10 4 cells per well. Next day, medium was removed and the 96-well plates were replaced with media containing the serially diluted compounds and the cells were further incubated for 48 h. The culture medium was removed and washed with 1× PBS. The next step was conducted by antiviral activity assay above described. To calculate the CC 50 values, the results were transformed to percentage of controls and the CC 50 values were graphically obtained from the dose-response curves. Ribavirin was used as positive, and dimethyl sulfoxide (DMSO) was used as negative control.
The effect of compound 1 or compound 3 on HRV-induced CPE was observed. Briefly, HeLa cells were seeded onto a 96-well culture plate at a concentration of 2 × 10 4 cells per well. The next day, the medium was removed and washed with PBS. Then, 0.09 mL of diluted virus suspension and 0.01 mL of medium supplemented with 20 mM MgCl 2 containing compound 1 or compound 3 of 20 μg/mL were added. After incubation at 32 °C in 5% CO 2 for 2 days, the morphology of cells was observed under microscope of 32 × 10 magnifications (St Ernst-Leitz, Wetzlar, Germany), and images were recorded.

Conclusions
Six polybromocatechols were isolated for the first time from a methanol extract of the red alga Neorhodomela aculeata. Two of these compounds, compounds 1 and 3, exhibited inhibitory activity against human rhinovirus activity in a HeLa cell line. Assuming that these compounds exhibit other drug-like properties, it will be interesting to investigate the preclinical and clinical efficacy of these polybromocatechols.