Cytotoxic and Antibacterial Angucycline- and Prodigiosin- Analogues from the Deep-Sea Derived Streptomyces sp. SCSIO 11594

Two new C-glycoside angucyclines, marangucycline A (1) and marangucycline B (2), along with three known compounds, dehydroxyaquayamycin (3), undecylprodigiosin (4) and metacycloprodigiosin (5), have been identified as products of the deep-sea sediment strain Streptomyces sp. SCSIO 11594. New structures were elucidated on the basis of HRESIMS, 1D and 2D NMR analyses and comparisons to previously reported datasets. Compounds 2 and 4 displayed in vitro cytotoxicity against four cancer cell lines A594, CNE2, HepG2, MCF-7 superior to those obtained with cisplatin, the positive control. Notably, compound 2 bearing a keto-sugar displayed significant cytotoxicity against cancer cell lines with IC50 values ranging from 0.24 to 0.56 μM; An IC50 value of 3.67 μM was found when using non-cancerous hepatic cell line HL7702, demonstrating the cancer cell selectivity of 2. Compounds 1–3 were proved to have weak antibacterial activities against Enterococcus faecalis ATCC29212 with an MIC value of 64.0 μg/mL. Moreover, 3 displayed selective antibacterial activity against methicillin-resistant Staphylococcus epidermidis shhs-E1 with an MIC value of 16.0 μg/mL.


Cytotoxicities and Antibacterial Activities
The angucyclines are a large group of natural products; members are characterized by an angular tetracyclic (benz[α]anthracene) structure with a hydrolysable sugar moiety. Angucyclines often express a broad range of biological activities. Members of the angucyclines have been noted as potent cytotoxins, antibacterials, antivirals and as inhibitors of assorted enzymes and of platelet aggregation [17,21]. The first reported compounds of this class were tetrangomycin and tetrangulol [17]. To our knowledge, the sugar unit of these species was usually linked at the tetrangomycin C-9 with a C-C bond and at the tetrangulol C-8 with a C-O bond. The antitubercular and cytotoxic compound dehydroxyaquayamycin (3), was the first compound shown to have a C-9 linked sugar unit using C-C connectivity with the tetrangulol skeleton. This agent was obtained as a derivative of aquayamycin [15] and later isolated as a natural product from the marine-derived Streptomyces sp. BCC45596 [14]. Compounds 1 and 2 were additions to this class of natural products; 1-3 were isolated from marine-derived Streptomyces sp. SCSIO 11594 as secondary metabolites.
Compounds 1-5 were tested for potential in vitro cytotoxicity against human lung cancer cell line A549, human nasopharyngeal carcinoma cell line CNE2, human breast adenocarcinoma cell line MCF-7, human hepatocarcinoma cancer cell line HepG2, and the normal hepatic cell line HL7702. The results were shown in Table 2. These data indicate that compound 4 is ≈1-10-fold more cytotoxic than the positive control cisplatin whereas compound 2, with its keto-sugar moiety, is ≈10-20-fold more potent than cisplatin. Interestingly, 2 and 4 demonstrate significant cytotoxic selectivity, with estimated therapeutic ratio values of 7-5 and 3-45, respectively, as reflected by comparisons of tumor cell-derived IC50 values and those obtained using HL7702 cells (non-cancerous control). Detailed examination of the structure-activity relationship (SAR) of the cytotoxicities of compounds 1-3 revealed that (i) the presence of ketone group and C-2″-O-C-3′ connection in the disaccharide moiety of compound 2 is critical important for the cytotoxicity; and (ii) the appearance of α-amicetose in the disaccharide chain sharply decreased the cytotoxicity. The SAR study of compounds 4 and 5 revealed that the open form of the aliphatic side chain plays an important role for the cytotoxicity. The anticancer activity of the related natural products has been described in a number of literatures [22][23][24], and the structure activity relationships of related synthetic natural products have been reported [25][26][27][28].
The antibacterial activities of compounds 1-5 were evaluated using MRSE shhs-E1, methicillin-resistant Staphylococcus aureus (MRSA) shhs-A1, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC29212, Escherichia coli ATCC 25922, Micrococcus luteus, and multidrug resistant Pseudomonas aeruginosa. These assays revealed that 1-3 have weak antibacterial activity against Enterococcus faecalis ATCC29212 with a MIC of 64.0 μg/mL in all cases and that 3 is selective against MRSE shhs-E1 demonstrating an MIC of 16.0 μg/mL. Compounds 4 and 5 did not show antibacterial activities against any of the above tested bacteria at a concentration up to 64.0 μg/mL.

Identification of Strain SCSIO 11594
Strain SCSIO 11594 (Figure 4a) was isolated from a sediment sample collected from the South China Sea (115°27.751 E, 19°28.581 N) at a depth of 2403 m using HRA medium (histidine 0.1 g, raffinose 1.0 g, Na2HPO4 0.3 g, KCl 1.7 g, MgSO4·7H2O 0.05 g, FeSO4·7H2O 0.01 g, CaCO3 0.02 g, agar 12 g, pH7.2, seawater 500 mL, distilled water 500 mL) with incubation at 28 °C for up to 4 weeks. The strain is preserved at the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences. Extraction of genomic DNA, PCR amplification, sequencing of the 16S rRNA gene, and phylogenetic analysis were performed as described by You and co-workers [29]. The 16S rRNA gene sequence has been deposited in GenBank with accession no. KP276583. The results of phylogenetic analyses showed that strain SCSIO 11594 should be a member of the genus Streptomyces. The 16S rRNA gene sequence of strain SCSIO 11594 demonstrated the highest similarity value to Streptomyces rubrogriseus LMG 20318 T (99.86%) and was found to cluster with members of the genus Streptomyces in the phylogenetic tree (Figure 4b). (a)

Fermentation, Extraction and Isolation of the Compounds
The strain was inoculated to a modified ISP-4 agar plate (soluble starch 1.0%, K2HPO4 0.1%, MgSO4·7H2O 0.1%, (NH4)2SO4 0.2%, CaCO3 0.2%, sea salt 3.0%, pH 7.2 before sterilization) from a glycerol tube under aseptic conditions and incubated 5 day at 28 °C . The mycelium was then transferred into 250 mL Erlenmeyer flasks each containing 50 mL of modified-AM2ab medium (soluble starch 0.5%, soybean powder 0.5%, yeast exact power 0.2%, bacterial peptone 0.2%, glucose 2.0%, KH2PO4 0.05%, MgSO4·7H2O 0.05%, NaCl 0.40%, sea salt 3.0% CaCO3 0.2%, pH 7.2 before sterilization) and incubated on rotary shakers with 200 rpm at 28 °C for 36 h. Each culture (seed) was then inoculated into 1 L Erlenmeyer flasks containing 200 mL of modified-AM2ab and fermentation continued under the conditions used to generate the seed cultures. Incubation was carried out under these conditions for 8 day. The 16 L of culture was then harvested and centrifuged at 3214× g (3900 rpm) for 10 min to yield the supernatant and mycelial cake, which was extensively extracted by butanone and acetone, respectively. The extracted residues were combined after HPLC analyses to validate extract contents.
The combined residues were subjected to silica gel CC using gradient elution initially using petroleum ether (

Cytotoxicity Assays
Compounds 1-5 were evaluated for cytotoxic activity using four human cancer cell lines, A549, CNE2, HepG2, MCF-7, one normal hepatic cell line HL7702 and previously reported MTT methodologies [30]. IC50 values were calculated using GraphPad Prism 5 software. All data were obtained in triplicate and are presented as means ± SD. Cisplatin was used as a positive control.

Antibacterial Activities Assay
The antibacterial activities of compounds 1-5 were assessed using seven strains of pathogenic bacteria including MRSE shhs-E1, MRSA shhs-A1, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC29212, Escherichia coli ATCC 25922, Micrococcus luteus, and multidrug resistant Pseudomonas aeruginosa. Dilution antimicrobial susceptibility tests for aerobic bacteria were carried out as previously reported [31,32]. Lowest concentrations of antimicrobial agents that completely inhibit cell growth in microdilution wells were determined by naked eye.

Conclusions
Five compounds including two new angucycline antibiotics, marangucycline A (1) and marangucycline B (2), were isolated from the deep-sea derived Streptomyces sp. SCSIO11594. Compounds 2 and 4, especially the new C-glycoside angucycline compound 2, displayed in vitro cytotoxicities against four cancer cell lines A594, CNE2, HepG2, MCF-7 superior to those noted with cisplatin. Remarkably, compounds 2 and 4 demonstrated significant anti-tumor selectivity. These data enable important correlations of structure to biological function and may be important for future drug lead efforts. Additionally, dehydroxyaquayamycin (3) was found to exert selective antibacterial activity against MRSE shhs-E1. This realization may prove important during the course of structure-activity relationship studies aimed at new antibacterial drug discovery/design studies.