Zhaoshumycins A and B, Two Unprecedented Antimycin-Type Depsipeptides Produced by the Marine-Derived Streptomyces sp. ITBB-ZKa6

Marine actinomycetes are prolific chemical sources of complex and novel natural products, providing an excellent chance for new drug discovery. The chemical investigation of the marine-derived Streptomyces sp. ITBB-ZKa6, from Zhaoshu island, Hainan, led to the discovery of two unique antimycin-type depsipeptides, zhaoshumycins A (1) and B (2), along with the isolation of the four known neoantimycins A (3), F (4), D (5), and E (6). The structures of the new compounds 1 and 2 were elucidated on the basis of the analysis of diverse spectroscopic data and biogenetic consideration. Zhaoshumycins A (1) and B (2) represent a new class of depsipeptides, featuring two neoantimycin monomers (only neoantimycin D or neoantimycins D and E) linked to a 1,4-disubstituted benzene ring via an imino group. Initial toxicity tests of 1–6 in MCF7 human breast cancer cells revealed that compounds 5 and 6 possess weak cytotoxic activity. Further structure–activity relationship analysis suggested the importance of the NH2 group at C-34 in 5 and 6 for cytotoxicity in MCF7 cells.


Introduction
Marine-derived actinomycetes are an excellent treasure house of structurally novel and biologically active natural products with potent medicinal properties [1][2][3][4][5]. In recent years, a various complex secondary metabolites, exemplified by the recently reported atratumycin, lobophorin, angucycline glycosides, tetrahydroisoquinolines, and polycyclic tetramate macrolactams, were isolated from marine-derived Streptomyces strains residing on or in mollusks, algae, corals, and sediments, indicating the marine-derived genus Streptomyces as a predominant source of chemically diverse and bioactive natural products [6][7][8][9][10]. Antimycins are a family of depsipeptide compounds typically discovered in actinomycetes, displaying a broad range of promising biological activities including anticancer, antiviral, antifungal, and nematocidal effects [11]. They are generated by a hybrid multimodular protein complex of non-ribosomal peptide synthetase and polyketide synthase assembly lines and have a common core structure composed of a macrocyclic ring with an amide linkage to a 3-formamidosalicylate unit [12][13][14]. This kind of depsipeptide natural products have attracted researchers' attention due to their diverse structures and important bioactivities. Structurally, members of the antimycin family differ by the varying size of their macrocyclic ring and modifications of their ester forming α-hydroxy acid components. So far, four major antimycin-type depsipeptides that differ by ring size, including 9-, 12-, 15-, and 18-membered macrocyclic ring-bearing antimycins, have been reported.
In our continuous work to exploit structurally novel and bioactive natural products from actinomycetes living in underexplored ecological niches [15][16][17][18], a marine-derived actinomycete strain identified as Streptomyces sp. ITBB-ZKa6 was investigated. Examining the fermentation extract of this strain, we discovered the new antimycin-type depsipeptides 1 and 2. Herein, we report the isolation, structure elucidation, and cytotoxic activities of zhaoshumycins A (1) and B (2) from this strain.

Results
The producing strain ITBB-ZKa6 was isolated from a marine sample obtained in Zhaoshu island, Sansha, Hainan at a depth of 1 m. We performed 16S rRNA sequence analysis that showed it to be similar to Streptomyces species. HPLC analysis of the fermentation extracts of this strain revealed an abundant metabolite profile in medium F, showing a series of compounds with complex UV/VIS absorptions. Subsequent large-scale fermentation of Streptomyces sp. ITBB-ZKa6 in medium F, followed by extraction of the cultures with EtOAc, chromatographic purification including silica gel, ODS, Sephadex LH20 chromatography, and semi-preparative HPLC, resulted in the discovery of compounds 1-6 ( Figure 1). The known neoantimycin A (3), neoantimycin F (4), neoantimycin D (5). and neoantimycin E (6) were characterized by comparing their MS and NMR spectroscopic data with those reported (Figures S16-S19, Tables S1-S4) [12,13,19].
Compound 1 was isolated as a yellow amorphous powder. High-resolution electrospray ionization mass spectrometry analysis (    10.9/0.88). Interestingly, the 13 C NMR data accounted for only half of the carbons observed in the molecular formula of 1, indicating its symmetry in the structure. The 1 H and 13 C NMR data of 1 were almost identical to those of neoantimycin D (5), except for the presence of two downfield-shifted carbon resonances of C-33 and C-34 in the 1,2,3-trisubstituted benzene ring of 1, and extra NMR data attributed them to a 1,4-disubstituted benzene ring.
Compound 2 was obtained as a yellow amorphous powder. The molecular formula of 2 was determined to be C 75 H 92 N 4 O 22 , as deduced by its HRESIMS analysis (observed an [M + Na] + ion peak at m/z 1423.6092, calcd 1423.6095) ( Figure S25). The NMR and UV spectra were very similar to those of 1, indicating they share a similar core structure. Comparing the 1 H ( Figure S9), 13 C (Figure S10), and DEPT135 ( Figure S11) NMR data of 2 ( Table 2) Figure 2) allowed its planar structure to be determined as shown; the compound was named zhaoshumycin B.
The relative stereochemistry of 1 and 2 was established by NOESY ( Figure S7 and Figure S15) NMR spectrum analysis, which implied that 1 and 2 have the same configurations as those reported for neoantimycin A-F [12,13]. The optical rotation of 1 (+100, c 0.1, MeOH) and 2 (+90, c 0.1, MeOH) were also similar to those of neoantimycin D (5) and E (6) [12], which were co-isolated from the extract. Taking this into consideration, the absolute structures of 1 and 2 were assigned, as shown in Figure 1, on the basis of a common biosynthetic origin shared with 5 and 6.
Antimycin-type natural products have been reported to possess promising anticancer activities against a panel of human cancer cell lines [14]. To obtain preliminary information of the cytotoxic activity of compounds 1-6, we assessed their toxicity toward the MCF7 human breast cancer cell line. Only compounds 5 and 6 showed activity at a concentration of 50 µM, with an inhibition rate of 91.08% and 90.53%, respectively. However, no obvious activity was observed for compounds 1-4 under our experimental conditions. To further understand the structure-activity relationship, we speculated that the loss of toxicity for compounds 1-4 in MCF7 cells could be potentially attributed to the derivatization of the NH 2 group at C-34, clearly signifying its importance in biological activity.

Strain Isolation and Identification
The actinomycete strain ITBB-ZKa6 was isolated by Z.G. from a marine sediment sample near a marine invertebrate, a yellow sponge that lived 1 m underwater, collected in Zhaoshu island, Sansha, Hainan, P.R. China. It was identified as a member of the Streptomyces genus by its morphological characteristics and 16S rRNA sequence analysis and comparison with other reported sequences in the NCBI GenBank database. The 16S rRNA sequence of Streptomyces sp. ITBB-ZKa6 was deposited in GenBank (accession no. OK381602), and this strain is preserved in the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

Cytotoxic Activity Test
The cytotoxic activity of compounds 1-6 was in vitro evaluated using the human breast cancer cell line MCF7 (purchased from the Jiangsu Provincial Center for Disease Prevention and Control) according to the previously reported MTT method [20]. Dimethyl sulfoxide (DMSO) was used to dissolve the tested compounds.

Conclusions
During the analysis of novel natural products from the fermentation extract of actinomycetes from underexplored ecological niches, we discovered two unique dimeric antimycin-type depsipeptides, zhaoshumycins A (1) and B (2), together with the four known neoantimycins A (3), F (4), D (5), and E (6). These compounds were obtained from the marine-derived Streptomyces sp. ITBB-ZKa6 from Zhaoshu island, Hainan. Zhaoshumycins A (1) and B (2) were identified as a new class of depsipeptides, featuring two neoantimycin moieties (only neoantimycin D or neoantimycins D and E) linked to a 1,4-disubstituted benzene ring via two imino groups. To the best of our knowledge, Zhaoshumycins A (1) and B (2) are the first examples of dimeric antimycin-type depsipeptides found in nature, representing a new class of this kind of natural products. This new structural feature expands the structural diversity of antimycin-type depsipeptides. A preliminary in vitro cytotoxicity test of 1-6 in MCF7 human breast cancer cells revealed that only compounds 5 and 6 possess a weak cytotoxic activity, indicating the NH 2 group at C-34 in 5 and 6 could be essential for cytotoxic activity in MCF7 cells. The discovery of 1 and 2 highlights the potential of marine actinomycetes as a prolific source of novel natural products for drug discovery.