A New Analogue of Echinomycin and a New Cyclic Dipeptide from a Marine-Derived Streptomyces sp. LS298

Quinomycin G (1), a new analogue of echinomycin, together with a new cyclic dipeptide, cyclo-(l-Pro-4-OH-l-Leu) (2), as well as three known antibiotic compounds tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated from Streptomyces sp. LS298 obtained from a marine sponge Gelliodes carnosa. The planar and absolute configurations of compounds 1 and 2 were established by MS, NMR spectral data analysis and Marfey’s method. Furthermore, the differences in NMR data of keto-enol tautomers in tirandamycins were discussed for the first time. Antibacterial and anti-tumor activities of compound 1 were measured against 15 drug-sensitive/resistant strains and 12 tumor cell lines. Compound 1 exhibited moderate antibacterial activities against Staphylococcuse pidermidis, S. aureus, Enterococcus faecium, and E. faecalis with the minimum inhibitory concentration (MIC) values ranged from 16 to 64 μg/mL. Moreover, it displayed remarkable anti-tumor activities; the highest activity was observed against the Jurkat cell line (human T-cell leukemia) with an IC50 value of 0.414 μM.


Introduction
With the emergence of newer resistant forms of infectious diseases and multi-drug resistant (MDR) bacteria and tumors, it has become essential to develop novel and more effective antibiotics [1]. In recent years, numerous studies have discovered that marine-derived actinomycete strains, mainly Streptomyces species, have the ability to produce a wide variety of biologically active and structurally unique metabolites. Some of these compounds possess strong antibacterial and anti-tumor activities [2][3][4]. The immense diversity of marine actinomycetes, along with their underutilization, has attracted great attention from researchers to discover novel antibiotics [5][6][7][8].
The strain LS298 was obtained from a marine sponge Gelliodes carnosa collected from the South China Sea. Based on the 16S rRNA sequence (GenBank accession number FJ937945) analysis [9] and the morphology, this strain was preliminarily identified as Streptomyces sp. Our previous studies have shown that the secondary metabolites of this strain contain echinomycin, cyclic dipeptides, and esters [10]. Among these compounds, echinomycin, a bifunctional DNA intercalator, is the predominantly and biologically active constituent against the Gram-positive and Gram-negative bacteria and also shows good anti-tumor activity [11][12][13][14]. Our continued search for echinomycin analogues or other novel antibiotics from extracts of large scale fermentation led to the isolation of two new compounds quinomycin G (1) and cyclo-(L-Pro-4-OH-L-Leu) (2), as well as three known compounds tirandamycin A (3), tirandamycin B (4), and staurosporine (5) (Figure 1). Structurally, quinomycin G (1) possessed a terminal double bond in one of the Ser groups. Cyclo-(L-Pro-4-OH-L-Leu) (2) was a new cyclic dipeptide. Tirandamycin A (3) was the 1-enol-4′-keto form, while tirandamycin B (4) was 1-keto-4′-enol form. It is the first time to reveal this form of tirandamycin B explicitly. In addition, antibacterial and anti-tumor activities of compound 1 were evaluated against 15 drug-resistant/sensitive strains and 12 tumor cell lines.

Structure Elucidation of Compounds 1-5
Quinomycin G (1) was obtained as an amorphous yellow powder, a molecular formula of C51H64N12O12S2 was determined by HRESIMS (m/z 1101.4288 [M + H] + , calcd for C51H65N12O12S2, 1101.4286), requiring 26 degrees of unsaturation. The chemical structure of 1 was adumbrated as an echinomycin analogue by the close similarity of its molecular formula and ultraviolet spectral properties (λmax (log ε) 245.2 nm (2.6), 325.8 nm (1.9), respectively) to those of echinomycin [10]. The 1 H NMR spectrum of 1 (Table 1) Figure S8) and HSQC of compound 1, indicated that compound 1 was comprised of two quinoxalines and eight amino acid moieties (two N-Me-Val, two Ala, two N-Me-Cys, one Ser, and one Dehydroxy-Ser) ( In the HMBC spectrum, the methylene protons (δH: 6.90 (1H, brs), 6.11 (1H, brs)) to C=O (δC: 163.2), confirmed that the double bond originated from the Ser. On the basis of the above information, all protons and carbon resonances were assigned and the planar structure of compound 1 was established. Because the planar differences in the structures of compound 1 and echinomycin cause the changes on spatial configurations, the NMR spectral data, especially 1 H NMR spectral data of compound 1 were different with that of echinomycin. The appearance of double bond of Dehydroxy-Ser may make the quinoxaline, amide, alkene, and carbonyl groups form a large conjugate plane (Supplementary Materials Figure S9). The CH3 of the Ala′ positioned in the shielding area, so its 1 H NMR spectral data upfielded to δH: 0.19.  Marfey's method was employed to assign the absolute configurations of the amino acid residues resulting from acid hydrolysis of 1 [15,16]. The 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) derivatives of the acid hydrolysate of 1 and the authentic D-and L-amino acids were subjected to HPLC analysis. The absolute configurations of all amino acid residues in 1 except for N-Me-Cys were established by comparing their HPLC retention times with those of the corresponding authentic D-and L-amino acid standards ( Table 2  Thus, as shown in 1 (Figure 1), the absolute stereochemistry of this novel echinomycin analogue was assigned and it was given the trivial name quinomycin G.
Subsequently, this inspired us to study the structural distinction between them. The literature survey indicated that the substituent groups on bicyclic ketal moiety have little influence on the NMR spectral data of the long conjugated system [17][18][19][20][21][22]. Therefore, we proposed that the distinct differences of NMR spectral data were caused by the positions of the enolic hydroxy and carbonyl group. Compared with C-1 at δC: 173.5 in tirandamycin A (3), the data of C-1 in tirandamycin B (4) moved to the downfield at δC: 181.0, implying that 4 should be in 1-keto-4′-enol form. The keto-enol tautomer existed widely in the structure of natural products, and the rules of NMR data of these two tautomers have been studied [23], which also supported that tirandamycin B (4) was the 1-keto-4′-enol form. It is the first time to reveal the 1-keto-4′-enol form of tirandamycin B explicitly. Because the structures of 1-keto-4′-enol form of tirandamycins were unclear previously, the assignments of the NMR data of these compounds were not correct [21,22]. Herein, we summarized the trend in NMR data of keto-enol tautomers exist in tirandamycins in order to raise concern on the structural and NMR data differences between these two forms. In the 13 C NMR spectrum, when the structure was in 1-enol-4′-keto form just like tirandamycin A, the carbon signals occurred at approximately δC: 173.5 (C-1), 116.2 (C-2), 147.9 (C-3) and 143.7 (C-5), however the carbons of the 1-keto-4′-enol form as tirandamycin B resonated at approximately δC: 181.0 (C-1), 124.8 (C-2), 143.2 (C-3) and 137.9 (C-5). More importantly, three olefinic protons had obvious differences in these two tautomers in the 1 H NMR spectrum, δH: 7.05 (H-2) 7.47 (H-3) and 6.19 (H-5) in 1-enol-4′-keto form changed to δH: 7.55 (H-2) 7.14 (H-3) and 5.81 (H-5) in 1-keto-4′-enol form. The chemical shift of H-2 at δH: 7.55 increased abnormally, which may be due to the shielding effect of the double bond (1C=O). According to the above results and literature survey [17][18][19][20][21][22]24], we also summed up a brief rule that if the 1 H NMR data of H-5 is more than δH: 6.00, the structure of tirandamycin is in 1-enol-4′-keto form, otherwise, it is in the other form.   (4) were employed for studying the tautomerizm of keto-enol tirandamycins and the test temperatures were set at 40, 60, 80 °C. With the increase of test temperature, the structure of tirandamycin A (3) was still in the 1-enol-4′-keto form (Supplementary Materials Figure S21), but the structure of tirandamycin B (4) gradually transformed to 1-enol-4′-keto form (Supplementary Materials Figure S26). The results suggested that in the DMSO-d6 solution, tirandamycin A (3) was stable in 1-enol-4′-keto form, while tirandamycin B (4) was more stable in 1-keto-4′-enol form than the other form. The reason may be the structure itself or external factors, which need to be further investigated.
The known antibiotic staurosporine (5) was characterized by comparison of the respective spectral data (MS, 1 H, 13 C NMR) with those found in the literature [25].

Biological Assays
The novel echinomycin analogue compound 1 was assayed for antibacterial activities against  (Table 6).

Bacterial Material and Fermentation
The producing strain LS298 was isolated from a sponge Gelliodes carnosa collected from Lingshui Bay, Hainan Province of China near Xincun Harbor (18°24′5.49″ N, 109°59′37.76″ E), in August 2007 [9]. It was identified as Streptomyces sp. on the basis of the morphology and 16S rRNA gene sequence analysis by comparison with other sequences in the GenBank database. The DNA sequence was deposited in GenBank (Accession No. FJ937945). The strain LS298 was first cultivated on Gause I agar plates (Gause I: starch 20 g; KNO3 1 g; NaCl 0.5 g; K2HPO3 0.5 g; MgSO4 0.01 g; Natural seawater 1 L; pH 7.0-7.2) at 28 °C for three days. Then, the mycelia were inoculated into 500 mL Erlenmeyer flasks, each containing 100 mL of liquid A1 medium (A1: starch 10 g; Yeast extract 4 g; Peptone 2 g; Natural seawater 1 L; pH 7.0-8.0). The flasks were incubated at 28 °C on a rotary shaker (200 rpm) for three days. Seed culture (10 mL) was transferred into three hundred 500 mL Erlenmeyer flasks (each Erlenmeyer flask contained 100 mL A1 medium) and incubated at 28 °C on a rotary shaker (200 rpm) for nine days.

Hydrolysis of Compounds 1-2 and HPLC Analysis by Marfey's Method
Compounds 1 (1.0 mg) and 2 (1.4 mg) were dissolved in 6 N HCl (1 mL), and heated at 110 °C for 18 h. After cooling to room temperature, the hydrolysates were dried under reduced pressure and resuspended into 100 μL of H2O.Then they were treated with 1 M NaHCO3 (25 μL), and reacted with 100 μL of 1% (w/v) FDAA in acetone at 40 °C for 1.5 h. After cooling to room temperature, the mixture was added with 1 M HCl (25 μL) to neutralize and terminate the reaction. MeOH was then added to the quenched reaction to afford a total volume of 500 μL; 10 μL of each hydrolysate derivatization reaction was used for HPLC analysis using an Agilent C18 column (150 × 4.6 mm, 5 μM) with a solvent gradient from 15% to 45% solvent B (solvent A: CH3COOH/H2O, 0.05/99.95, solvent B: CH3CN) over the course of 30 min and UV detection at 340 nm at a flow rate of 1 mL/min. Similarly, 10 μL of the standard amino acids in H2O (4 μM) were added to 1 M NaHCO3 (20 μL) and each mixture was treated with 1% (w/v) FDAA (50 μL) for 1.5 h at 40 °C. Derivatization reactions were terminated with 1 M HCl (20 μL) and diluted to a total volume of 500 μL with MeOH. Of these standard amino acid derivatization reactions, 10 μL was subjected to HPLC analysis and used as structural standards in the elucidation of structures 1 and 2.

Biological Assays
Antibacterial and anti-tumor assays were performed with compounds of purity >90% by HPLC. (VSE), 09-9 (VRE)), which included strains from the ATCC collection and clinical isolates. MIC values against the 15 bacterial strains for compound 1 were measured by using the agar dilution method described by the Clinical Laboratory Standards Institute [26]. Briefly, the test medium was Mueller-Hinton broth, and the inoculum was 10,000 colony forming units (CFU)/spot. The compound 1 was incorporated into the agar medium, with each plate containing a different concentration of the compound. Culture plates were incubated at 35 °C for 18 h, and MICs were then recorded. The positive controls were levofloxacin and echinomycin. The final concentrations of compounds ranged from 0.03 to 128 μg/mL. The MIC was defined as the lowest concentration that prevented visible growth of the bacteria [27].
The human colonic carcinoma (HCT-116), human hepatoma (HepG2), human gastric cancer (BGC-823), human non-small cell lung cancer (NCI-H1650), human ovarian cancer (A2780), human pancreatic cancer (SW1990, Mia-PaCa-2), human multiform glioblastoma (U87 MG), human neuroblastoma (SK-N-SH), human renal clear cell carcinoma (ACHN, 786-O) were maintained in DMEM medium; human T-cell leukemia (Jurkat) was maintained in RPMI 1640 medium. Both media were supplemented with 10% heat inactivated fetal bovine serum, 100 units/mL of penicillin and 100 μg/mL of streptomycin, in a humidified 5% CO2/air atmosphere at 37 °C. MTT assay: briefly, logarithmic cells were digested with 0.25% pancreatic enzyme-EDTA and plated in the 96-well plates at concentration of 800-2000/100 μL per well. Compounds at final concentrations of 0.5 to 50 μg/mL were added with triplicates of each concentration after 24 h. The cells were incubated further at 37 °C for 96 h, the medium was aspirated, and 100 μL MTT of 0.5 mg/mL in medium was added. After 4h incubation, the medium was aspirated and 200 μL DMSO was added to solubilize the formazan crystals. Absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 650 nm. The dose-response curves were fitted with Sigma plot and IC50s were determined.

Conclusions
In summary, a novel echinomycin analogue quinomycin G (1), a new cyclic dipeptide cyclo-(L-Pro-4-OH-L-Leu) (2), along with three known antibiotics tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated and characterized from the marine Streptomyces sp. LS298. To our knowledge, this was the first time to obtain three types of antibiotics from one strain from the same batch, though these types of antibiotics have been isolated from different strains of genus Streptomyces [18,32]. What is more, the 1-keto-4′-enol form of tirandamycin B was reported firstly, and the trend in NMR data of keto-enol tautomers in tirandamycins was discussed.
Compound 1 exhibited moderate antibacterial and remarkable anti-tumor activities; however, its activities were lower than those of echinomycin, which indicated that the bicyclic peptide of these compounds was required for their activities. Echinomycin has been studied for many years and the mechanism of its antibacterial and antitumor activities is considered to be DNA bis-intercalation. Due to the similar structure, we proposed that compound 1 has a similar mechanism against the bacteria and the tumor cells. Efforts are underway to discover novel and potential echinomycin analogues in future work through combinations of genome mining and heterologous expression approaches.