Antiplasmodial activity of bacilosarcin A isolated from the octocoral-associated bacterium Bacillus sp. collected in Panama.

AIM: This study was designed for isolating and characterizing antiplasmodial compounds from marine octocoral-associated bacteria.
MATERIALS AND METHODS: The organic extract of the Bacillus sp. was subjected to purification using several chromatography techniques guided by bioassays to yield three isocoumarin derivatives (1-3). Chemical structures of the compounds were elucidated on the basis of HRMS spectra and NMR spectroscopy. The antiplasmodial activity of the isolated compounds was evaluated in vitro against the chloroquine-resistant Plasmodium falciparum strain W2.
RESULTS: Isolated compounds were identified as bacilosarcin A (1), AI77-F (2), and AI77-H (3). Bacilosarcin A (1) displayed a low micromolar activity (IC(50) = 2.2 μM) against P. falciparum while compounds 2 and 3 showed no activity.
CONCLUSIONS: Bacilosarcin A was found to be responsible for the antiplasmodial activity observed in the crude extract obtained from the Bacillus sp.

Malaria continues to be an uncontrolled disease that affects 106 countries in tropical regions, and half of the world's population is at risk of infection. According to the 2010 World Malaria Report of the World Health Organization (WHO), in 2009 there were 225 million cases of malaria and approximately 781,000 deaths.[1]

Efforts to develop an effective vaccine to prevent malaria have not been successful, despite the fact that several vaccine candidates have reached phase I–II, and even phase III clinical trials.[23] Additionally, the development of drug resistance by malaria parasites creates a more complex scenario for the treatment of this disease.[45] Given this situation, there is an urgent need for finding new antimalarial compounds with new mechanisms of action.

Natural products have played an important role in the discovery of novel compounds for the treatment of malaria. Several plant metabolites, such as quinine and artemisinin, have been the classical drugs used to treat this disease. These plant-derived compounds have also inspired synthetic drugs with improved antimalarial properties, such as chloroquine and artemeter. Unfortunately, Plasmodiun falciparum has developed resistance to all current drugs used for the treatment of malaria, except for artemisinin derivatives (ACTs).[3-5] However, a decrease in the in vivo susceptibility of P. falciparum to ACT treatment has been observed.[6]

Although terrestrial plants have been the traditional source of antimalarial drugs; the oceans have also proven to be a rich source of antimalarials, with over 60 antiplasmodial compounds isolated from marine organisms prior to 2010.[78] Among marine organisms, bacteria are emerging as an important resource for drug discovery due to their outstanding capacity to biosynthesize active molecules.[910] Marine cyanobacteria,[78] and sediment-associated actinomycetes[11] represent promising new sources of antimalarial compounds, even though their biosynthetic capacity has been used primarily in oncology.[10]

As a part of our drug discovery program on marine organisms, we are exploring the diversity of bacteria associated with Panamanian marine invertebrates as sources for new bioactive compounds. With this goal, crude extracts and fractions from marine bacteria were screened in a panel of assays against cancer and neglected tropical diseases including malaria, Chagas’ disease, and leishmaniasis. Herein we report the antiplasmodial activity of bacilosarcin A (1), an isocoumarin derivative isolated from the extract of the bacterium Bacillus sp. associated with the octocoral Leptogorgia alba.

Materials and Methods

Optical rotations were measured with a Jasco P-2000 polarimeter. NMR spectra were acquired on Jeol Eclipse 400 MHz and Bruker Avance 600 MHz spectrometers and referenced to residual solvent 1H and 13C signals (δH 7.26, δC 77.0 for CDCl3 ; and δH 2.50, δC 39.5 for DMSO-d6 ). Low-resolution ESIMS spectra were acquired on Jeol LC-mate mass spectrometer, while high-accuracy mass measurements were obtained on Agilent 6230 mass spectrometer. The purification of the compounds was carried out on Agilent 1200 HPLC system equipped with a quaternary pump, a diode array detector, and a normal phase silica gel column (Phenomenex Sphereclone, 4.6 mm × 100 mm, 5 μm) at a flow rate of 1 mL/min.

Biological material collection and identification

The octocoral L. alba was hand collected using SCUBA at 10 m near Otoque Island, located on the Pacific Ocean side off the coast of Panama in August 2009. A piece of the coral was rinsed with autoclaved seawater to remove loosely attached bacteria, and a small portion (0.5 mL) of the coral mucus was inoculated directly on agar plates with the seawater-based nutrient medium (8 g of Noble agar, 500 mg of mannitol, 100 mg of peptone, rifampicin [5 μg/mL] in 1 L of natural sea water). Agar plates were taken to the laboratory and observed for bacterial isolation, at room temperature over a period of 1 month. Strain GL0033 was further isolated from the collection plate and successively replated until a pure strain was obtained.

The coral specimen was identified as L. alba based on its morphology and SEM micrographs of the coral sclerites following Breedy and Guzman.[12] The taxonomic identification of bacterial strain GL0033 was carried out using PCR amplification and 16 S rRNA gene sequencing. The NCBI BLAST search for the GL0033 sequence indicated that this strain was 100% similar to Bacillus sp. Reference specimens of the coral (GLOT-120209-02) and the bacterial strain (GL0033) are deposited at INDICASAT's CEDD.

Fermentation and extraction

Bacterium GL0033 was inoculated in six 2 L Erlenmeyer flasks, each containing 1 L of the culture broth (4 g of peptone, 2 g of the yeast extract, and 2 mL of glycerol in 1 L of natural seawater). Twenty grams of sterile Amberlite® XAD-16 resin was added to each Erlenmeyer flask with the culture medium. After 10 days of cultivation at room temperature (20°C) at 172 rpm, the resin was filtered, washed with deionized water, and eluted with methanol. The culture medium (6 L) was extracted with EtOAc (3 × 6 L). The organic extracts were combined and the solvents were removed under reduced pressure to yield 3.4 g of the crude extract.

Isolation of compounds

The crude extract (3.0 g) was fractionated using reverse phase C-18 solid phase extraction (SPE) cartridges, eluted with a step gradient of 20%, 40%, 60%, 80%, and 100% MeOH in water. The MeOH/water 60:40 SPE fraction showed 84% growth inhibition of the P. falciparum. The fraction was then subjected to HPLC purification on a normal phase silica gel column (Phenomenex Sphereclone, 250 × 4.5 mm, 1 mL/min) using the isocratic elution of 7:3 EtOAc/hexanes, over 70 mins to yield 0.8 mg of bacilosarcin A (1), 0.7 mg of AI77-F (2), and 0.8 mg of AI77-H (3).

Bacilosarcin A (1): White amorphous powder, [α]2524.5 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 10.68 (1H, bs, OH), 7.42 (1H, dd, J = 8.2, 7.5 Hz, H-6), 6.94 (1H, d, J = 9.9 Hz, H-6’), 6.89 (1H, d, J = 8.2 Hz, H-7), 6.70 (1H, d, J = 7.5 Hz, H-5), 6.41 (1H, bs, NH), 4.59 (1H, dt, J = 13.1, 2.7 Hz, H-3), 4.39 (1H, m, H-5’), 4.09 (1H, dd, J = 9.9, 2.6 Hz, H-9’), 3.93 (1H, d, J = 9.9 Hz, H-8’), 3.50 (1H, dd, J = 7.0, 2.6 Hz, H-10’), 3.08 (1H, dd, J = 16.1, 13.1 Hz, H-4a), 2.84 (1H, dd, J = 16.1, 2.7 Hz, H-4b), 2.63 (1H, d, J = 17.9 Hz, H-11’a), 2.46 (1H, dd, J = 17.9, 7.0 Hz, H-11’b), 1.76 (1H, ddd, J = 13.8, 9.9, 4.9 Hz, H-4’a), 1.66 (1H, m, H-3’), 1.44 (ddd, J = 13.8, 9.1, 4.8 Hz, H-4’b), 1.34 (3H, s, H-13’), 1.30 (3H, s, H-16’), 0.97(3H, d, J = 6.4 Hz, H-2’), 0.95 (3H, d, J = 6.3 Hz, H-1’); 13C NMR (100 MHz, CDCl3) δ ppm 172.3 (C, C-7’), 171.3 (C, C-12’), 169.7 (C, C-1), 162.1 (C, C-8), 139.3 (C,C-10), 136.6 (CH, C-6), 118.2 (CH, C-5), 116.3 (CH, C-7), 108.1 (C, C-9), 98.0 (C, C-15’), 81.2 (CH, C-3), 72.5 (CH, C-9’), 69.7, (C, C-14’), 68.2 (CH, C-8’), 49.6 (CH, C-5’), 47.9 (CH,C-10’), 41.0 (CH2, C-4’), 30.5 (CH2, C-4), 30.3 (CH2, C-11’), 24.7 (CH, C-3’), 23.8 (CH3, C-16’), 23.1 (CH3, C-2’), 21.8 (CH3, C-1’), 21.3 (CH3, C-13’); HRESITOF-MS m/z 492.2338 [M + H]+ (calculated for C24H34N3O8, 492.2340).

AI77-F (2): White amorphous powder, [α]25 – 106.4 (c 0.2, CHCl3); 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.81 (1H, s, C8-OH), 7.83 (1H, d, J = 10.0, H-6’), 7.52 (1H, dd, J = 5.7, 1.1, H-10’), 7.50 (1H, t, J = 8.0 Hz, H-6), 6.86 (1H, d, J = 8.0, H-7), 6.84 (1H, d, J = 6.1 Hz, H-5), 6.28 (1H, dd, J = 5.7, 2.0, H-11’), 6.21 (1H, d, J = 6.0, C8’-OH), 5.36 (1H, dt, J = 3.8, 1.1, H-9’), 4.69 (1H, ddd, J = 8.9, 6.0, 4.2, H-3), 4.41 (1H, dd, J = 6.6, 3.8, H-8’), 4.17 (1H, ddd, J = 13.5, 10.0, 4.2, H-5’), 2.97 (2H, m, H-4), 1.69 (1H, m, H-4’-a), 1.58 (1H, m, H-3’), 1.31 (1H, ddd, J = 13.5, 10.0, 3.8, H-4’-b), 0.91 (3H, d, 6.6, H-2’), 0.84 (3H, d, 6.4, H-1’); 13C NMR (100 MHz, DMSO-d6) δ ppm 172.7 (C, C-12’), 169.7 (C, C-1), 168.8 (C, C-7’), 160.81 (C, C-8), 154.5 (CH, C-10’), 140.2 (C, C-10), 136.3 (CH, C-6), 121.7 (CH, C-11’), 118.5 (CH, C-5), 115.3 (CH, C-7), 108.4 (C, C-9), 84.1 (CH, C-9’), 80.8 (CH, C-3), 70.6 (CH, C-8’), 48.3 (CH, C-5’), 38.8 (CH2, C-4’), 29.1 (CH2, C-4), 24.05 (CH, C-3’), 23.3(CH3, C2’), 21.3 (CH3, C-1’); HRESIFT-MS [M + Na]+ m/z 412.1365 (calculated for C20H23NO7Na, 412.1367).

AI77-H (3): White amorphous powder, [α] 25 - 33 (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3) δ ppm 10.76 (1H, s, C8-OH), 7.66 (1H, d, J = 5.1 Hz, H-10’), 7.41 (1H, t, J = 7.9 Hz, H-6), 6.88 (1H, m, H-7), 6.87 (1H, m, H-6’), 6.69 (d, J = 7.9 Hz, H-5), 6.19 (1H, d, J = 5.1 Hz, H-11’), 5.30 (1H, d, J = 5.1 Hz, H-9’), 4.60 (1H, d, J = 13.6 Hz, H-3), 4.37 (1H, m, H-5’), 4.30 (1H, d, J = 5.2 Hz, H-8’), 3.02 (1H, dd, J = 16.1, 13.6 Hz, H-4a), 2.84 (1H, dd, J = 16.1, 2.6 Hz, H-4b), 1.83 (1H, m, H-4’a), 1.69 (1H, m, H-3’), 1.47 (1H, ddd, J = 13.8, 9.2, 4.6 Hz, H-4’b), 0.97 (3H, d, J = 6.6 Hz, H-2’), 0.95 (3H, d, J = 6.1 Hz, H-1’); 13C NMR (100 MHz, CDCl3) δ ppm 171.9 (C, C-12’), 169.4 (C, C-1), 168.9 (C, C-7’), 162.2 (C, C-8), 153.4 (CH, C-10’), 139.1 (C, C-10), 136.5 (CH, C-6), 122.9 (CH, C-11’), 118.2 (CH, C-5), 116.3 (CH, C-7), 108.0 (C, C-9), 83.3 (CH, C-9’), 80.8 (CH, C-3), 72.0 (CH,C-8’), 49.4 (CH,C-5’), 40.5 (CH2, C-4’), 30.3 (CH2, C-4), 24.7 (CH, C-3’), 23.0 (CH3,C-2’), 21.8 (CH3,C-1’); HRESIMS [M + Na]+ m/z 412.1369 (calculated for C20H23NO7Na, 412.1367).

Biological activity

The antiplasmodial activity was determined in a chloroquine-resistant P. falciparum strain (W2) utilizing a microfluorimetric assay to measure the inhibition of the parasite growth based on the detection of the parasitic DNA by intercalation with PicoGreen.[13] P. falciparum was cultured according to the methods described by Trager and Jensen.[14] The parasites were maintained in a 2% hematocrit on flat-bottomed flasks (75 mL) with the RPMI 1640 medium (Gibco-BRL) supplemented with 10% human serum.

Cytotoxicity was measured in MCF-7 human breast cancer cells with the cell viability being determined by MTT reduction.[15] Cells were seeded in 96-well plates at a rate of 6000 cells/well in 180 μL of the medium. After 24 h, the test chemicals were dissolved in DMSO and diluted in the medium without fetal bovine serum and then added to the plate at a rate of 20 μg/well. DMSO was less than 0.5% of the final concentration. After 48 h, the medium was removed and cell viability determined.

Results and Discussion

The bacterial strain GL0033 was isolated from the octocoral L. alba collected near Otoque Island, Gulf of Panama, Pacific Panama. The bacterium was identified as Bacillus sp. based on its 16S rRNA sequence.

The organic crude extract of strain GL0033 obtained from the culture broth showed a strong inhibition on the growing of chloroquine-resistant Plasmodium falciparum. The bioassay-guided fractionation of the extract using SPE fractionation followed by HPLC purification yielded compounds 1–3 [Figure 1]. Structures of 1–3 were identified as bacilosarcin A (1),[1617] AI77-F (2),[18] and AI77-H (3),[19] on the basis of their spectroscopic data (1H-NMR, 13C-NMR, HRESITOF-MS, and optical rotations) and their comparison with spectroscopic data from the literature.

Figure 1

Structures of compounds 1-3 isolated from Bacillus sp

Compounds 1–3 where evaluated for their activity against chloroquine-resistant P. falciparum [Table 1]. Bacilosarcin A (1) showed an IC50 value of 2.2 μM in the antiplasmodial assay, whereas compounds 1–2 did not show any activity [Table 1].

Table 1

Biological activity of compounds 1–3 against Plasmodium falciparum and MCF-7 breast cancer cell lines

Compounds 1–3 possess the same 3,4-dihydro-8-hydroxy-isocoumarin moiety; however, bacilosarcin A (1) includes a 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system attached to carbon C-8’, instead of the α-β-unsaturated butenolide ring present in compounds 2 and 3. From these structural features, we can infer that the heterobicyclic ring system attached to C-8’ in bacilosarcin A is required for its antimalarial activity. Compounds 1–3 were also evaluated for their cytotoxicity using MCF-7 human breast cancer cell lines and showed no significant cytotoxic effect in this assay [Table 1].

One of the more remarkable structural features of bacilosarcin A (1) is its unprecedented 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system which brought the attention of synthetic organic chemists resulting in the total synthesis of compound 1.[17]

This is the first report of the antiplasmodial activity of bacilosarcin A (1), which constitutes a new addition to the few existing antiplasmodial metabolites isolated from heterotrophic bacteria associated with corals. This finding confirms that bacteria associated with marine invertebrates represent a promising resource for antimalarial research.