Cytotoxic Anthracycline Metabolites from a Recombinant

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Cite This: J. Nat. Prod. 2018, 81, 1278−1289

Cytotoxic Anthracycline Metabolites from a Recombinant Streptomyces Chun Gui,†,‡ Jie Yuan,§ Xuhua Mo,⊥ Hongbo Huang,†,‡ Shanwen Zhang,†,‡ Yu-Cheng Gu,∥ and Jianhua Ju*,†,‡ †

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, People’s Republic of China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 110039, People’s Republic of China § Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510301, People’s Republic of China ⊥ Shandong Province Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China ∥ Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, U.K. S Supporting Information *

ABSTRACT: The C7 (C9 or C10)-O-L-rhodosamine-bearing anthracycline antibiotic cytorhodins and their biosynthetic intermediates were recently isolated from Streptomyces sp. SCSIO 1666. Cosmid p17C4 from the Streptomyces lydicus genomic library, which harbors both the biosynthetic genes for L-rhodinose (or 2-deoxy-L-fucose) and its glycosyltransferase (encoded by slgG), was introduced into SCSIO 1666 to yield the recombinant strain Streptomyces sp. SCSIO 1666/17C4. Chemical investigations of this strain’s secondary metabolic potential revealed the production of different anthracyclines featuring C7-O-L-rhodinose (or 2-deoxy-L-fucose) instead of the typically observed L-rhodosamine. Purification of the fermentation broth yielded 12 new anthracycline antibiotics including three new ε-rhodomycinone derivatives, 1, 4, and 8, nine new β-rhodomycinone derivatives, 2, 3, 5−7, and 9−12, and three known compounds, L-rhodinose-L-rhodinose-Lrhodinoserhodomycinone (13), ε-rhodomycinone (14), and γ-rhodomycinone (15). All compounds were characterized on the basis of detailed spectroscopic analyses and comparisons with previously reported data. These compounds exhibited cytotoxicity against a panel of human cancer cell lines. Significantly, compounds 4 and 13 displayed pronounced activity against HCT-116 as characterized by IC50 values of 0.3 and 0.2 μM, respectively; these IC50 values are comparable to that of the positive control epirubicin.

A

transferases (GTs), CytG1−G3, involved in the sugar transfer processes.12 Cytorhodins are type II PKS-derived metabolites whose synthesis appears to commence with a propionyl-CoA starter unit followed by condensation of nine malonyl-CoA units catalyzed by a minimal polyketide synthase (Ksα, KSβ, and ACP); the C-21 backbone ε-rhodomycinone is the direct result of the PKS machinery. Several sugar tailoring modifications lead to the mature products. Specifically, CytG1 is responsible for appending the first L-rhodosamine moiety at the C-7 position; CytG2 then transfers the second and third L-rhodinose (or 2deoxy-L-fucose) moieties to the trisaccharide chain, and CytG3 appears to transfer the L-rhodosamine to both the C-10 and C9 positions.

nthracycline antibiotics are cytotoxic to a variety of cancer cell lines and are widely used in the clinic.1 These metabolites are generally derived from actinomycetes and their genetically modified mutant strains.2 Examples of such actinomycete-derived agents include daunorubicin, doxorubicin, epirubicin, and aclacinomycin.3 Despite their clinical utility, serious side effects are often associated with the anthracyclines; cumulative cardiotoxicity and the development of multiple drug resistance4−6 are paramount among these side effects. These observations have inspired us to search for new anthracyclines with superior pharmacological properties. Our previous investigations of the tirandamycins7−11 and anthracycline antibiotic cytorhodins12 revealed that both could be readily isolated from Streptomyces sp. SCSIO 1666 and its mutant strain. We identified the biosynthetic gene cluster (BGC) of cytorhodin (cyt) and characterized three glycosyl© 2018 American Chemical Society and American Society of Pharmacognosy

Received: March 14, 2018 Published: May 16, 2018 1278

DOI: 10.1021/acs.jnatprod.8b00212 J. Nat. Prod. 2018, 81, 1278−1289

Journal of Natural Products

Article

Figure 1. Organization of (a) streptolydigin BGC from Streptomyces lydicus NRRL 2433; (b) cytorhodin BGC from Streptomyces sp. SCSIO 1666; (c) chemical structures of tirandamycins, streptolydigin, cytorhodins, and their intermediates. *p17C4 represents the cosmid containing all Lrhodinose biosynthesis genes along with the GT-encoding gene slgG and NRPS encoding genes.

Figure 2. HPLC analysis of the fermentation broth of mutant strain Streptomyces sp. SCSIO 1666/17C4 and wild-type strain Streptomyces sp. SCSIO 1666.

Streptomyces lydicus and contains all the L-rhodinose biosynthesis genes as well as the GT encoding gene slgG (Figure 1). Chemical investigation of the fermentation broth for Streptomyces sp. SCSIO 1666/17C4 revealed a diverse metabolite profile relative to the wild-type SCSIO 1666 strain (Figure 2). The newly formed peaks were characterized by UV profiles very reminiscent of anthracycline-like metabolites. Fifteen C7-O-L-rhodinose (or 2-deoxy-L-fucose)-bearing anthracyclines, including three new ε-rhodomycinone derivatives (1, 4, and 8), nine new β-rhodomycinone derivatives (2, 3, 5− 7, and 9−12), and three known compounds (L-rhodinose-L-

It is well documented that the sugar moiety is essential for anthracycline antibiotic’s antitumor activity. Even small modifications to this moiety significantly alter cytotoxic activities and, often, the modes of action for anthracyclines.13 Given this knowledge, we envisioned the application of combinatorial biosynthetic methods to generate glycosylated derivatives with altered pharmacological profiles. Here, we describe the introduction of cosmid p17C4 into the strain Streptomyces sp. SCSIO 1666 en route to the recombinant strain Streptomyces sp. SCSIO 1666/17C4. Importantly, the cosmid p17C4 is derived from the streptolydigin BGC14 from 1279



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Chart 1

Figure 3. Construction of mutant strain Streptomyces sp. SCSIO 1666/17C4.

rhodinose-L-rhodinose-rhodomycinone (13), ε-rhodomycinone (14), and γ-rhodomycinone (15)), were subsequently purified and structurally elucidated. In this paper, we describe hybrid strain construction, structure elucidation, and biological activity evaluations for these compounds.

elements necessary for conjugation. Using standard conjugation methods,15 p17C4-pSET152AB was introduced into the heterologous Streptomyces host, and its DNA locus integrated into the chromosome of Streptomyces sp. SCSIO1666. The exconjugator with the correct genotype was designated as Streptomyces sp. SCSIO 1666/17C4. Subsequent 15 L scale fermentation of the mutant strain afforded the crude extract containing all anthracycline congeners of interest. Silica gel chromatography and RP-HPLC purification methods afforded analytically pure samples of 1−15. Structure Elucidation. Compound 13 was isolated as a red, amorphous powder. HRESIMS afforded ion peaks at m/z 769.3081 [M − H]− and m/z 793.3034 [M + Na]+. Full

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RESULTS AND DISCUSSION Construction of Streptomyces sp. SCSIO 1666/17C4, Its Fermentation, and Isolation of Metabolites 1−15. For heterologous expression of cosmid p17C4 in Streptomyces sp. SCSIO1666 (Figure 3), the cosmid p17C4 was cloned into the plasmid pSET152AB15 downstream of ermE* to afford p17C4pSET152AB. The plasmid p17C4-pSET152AB contained all 1280



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Compound 3 was isolated as a red, amorphous powder. It was found to have the same molecular formula as compound 2 (C38H48O15) on the basis of HRESIMS. These two compounds also exhibited almost identical 1H and 13C NMR spectra. Careful analysis of these data revealed a key difference in each trisaccharide linkage order between 2 and 3. The trisaccharide chain in 3 was found to be L-rhodinose-L-rhodinose-2-deoxy-Lfucose. This differs significantly from the L-rhodinose-2-deoxyL-fucose- L-rhodinose trisaccharide found in 2. This key difference in trisaccharides was made clear only after comprehensive interpretation of full sets of 2D NMR data, especially COSY and HMBC correlations (Figure 5) for each compound. On the basis of these results and conclusions compound 3 was subsequently named L-rhodinose-L-rhodinose2-deoxy-L-fucose-β-rhodomycinone. Compound 4 was isolated as a red, amorphous powder, and its molecular formula established as C38H48O14 on the basis of HRESIMS. Similarities observed for both the 1H and 13C NMR data for 4 and 3 indicated that both compounds contained the same trisaccharide. The only distinct spectroscopic difference between 3 and 4 involved the presence of two coupled doublet signals (δH 2.57, J = 19.0 Hz; δH 3.26, J = 19.0 Hz) assigned as C-10-linked methylene protons in compound 4; this was in stark contrast to the previously noted C-10 methine of 3. Detailed analysis of the 2D NMR data (Figure 5) established the structure of 4 as L-rhodinose-L-rhodinose-2-deoxy-L-fucose10-decarbomethoxy-ε-rhodomycinone. Compound 5 was isolated as a red, amorphous powder, and its molecular formula established as C44H58O17 on the basis of HRESIMS. Notably, the ion peaks observed for this species revealed the clear presence of an additional C6H9O2 relative to the same data previously generated for trisaccharide 3. We reasoned that this may be attributable to an additional Lrhodinose moiety. Comparisons of 1H and 13C spectra for 5 versus compound 3 revealed the presence of four sugars in 5, although it was clear that they were contiguous and, thus, constituted a tetrasaccharide. Consistent with this logic, four anomeric carbons at δC 101.4, 100.4, 100.3, and 99.6 were also observed. Thus, the tetrasaccharide chain consisting of four Lrhodinose moieties attached in a manner highly similar to that seen with 3 was noted. Also noteworthy was that this tetrasaccharide was found to be attached to the aglycone in a fashion similar to that noted with 3. On the basis of these data and their relationships to compounds 1−4 we ultimately named 5 as L-rhodinose-L-rhodinose-L-rhodinose-L-rhodinose-β-rhodomycinone. Compound 6 was isolated as a red, amorphous powder. Its molecular formula was established as C38H48O16 on the basis of HRESIMS, which revealed that 6 contains one O atom more than compound 3. Comparisons of the 1H, 13C, and 2D NMR spectroscopic data for 6 with those of 3 (Table 1, Figure 5) revealed that 6 possesses an additional C-3″ OH substitution relative to 3; the methylene group at C-3″ (δH 1.73, δC 24.3) in 3 is replaced by an oxygen-bearing methine carbon (δH 4.11, δC 65.3) in 6. In short, sugar unit B (L-rhodinose) in 3 is hydroxylated to render 2-deoxy-L-fucose as the central sugar of the pendant trisaccharide in 6. This structural difference is consistent with changes observed for the neighboring C-4″ shift; in 3 the resonance for C-4″ is δC 75.0, but in 6 the same resonance is significantly deshielded to δC 82.8. Consistently, HMBC correlations from H-1″ and H-4″ to C-3″ as well as the COSY correlation of H-2″/H-3″ confirmed this substitution. Additionally, the three deoxysugar moieties were confirmed to

assignment of this compound (Table S1) revealed it to be the known compound L-rhodinose-L-rhodinose-L-rhodinose-ε-rhodomycinone (13), an ε-rhodomycinone derivative previously isolated from Strepomycetes sp. HPL Y-11472 with antibacterial activity against Micrococcus luteus with MIC values of 50 10.2 ± 1.0 40.2 ± 0.8 5.4 ± 0.9 23.9 ± 0.5 4.5 ± 0.2 13.2 ± 1.0 10.9 ± 0.4 0.9 ± 0.1

HCT-116 3.6 6.1 32.8 0.3 7.2 5.0 5.8 3.1 13.2 9.8 1.4 5.4 0.2 9.6 6.0 0.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 1.1 1.7 0.1 1.3 0.4 0.2 0.3 0.8 0.5 0.2 0.8 0.07 1.3 0.6 0.1

HepG2

MCF10A

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.6 ± 1.4 6.7 ± 1.3 5.2 ± 0.4 2.2 ± 0.3 5.7 ± 1.5 3.4 ± 1.6 10.5 ± 0.7 16.9 ± 1.6 2.5 ± 1.1 3.7 ± 0.3 2.0 ± 0.3 10.4 ± 1.4 1.4 ± 0.4 7.1 ± 0.03 11.9 ± 1.2 0.1

6.4 5.7 5.5 8.6 8.5 1.5 6.2 11.3 2.0 8.0 0.9 9.3 1.7 13.5 13.0 0.1

1.0 0.5 1.6 0.1 2.4 1.3 0.2 2.4 0.4 1.6 0.1 1.0 0.5 2.3 1.5 0.01

Values are the average of three independent replicates. Epirubicin was used as positive control. IC50 in μM. “ND”, not detected/determined. lines were carried out according to previously described methods.17 The detailed process is described in the Supporting Information.

L -Rhodinose-2-deoxy-L -fucose-2-deoxy- L -fucose-β-rhodomycinone (6): red powder; [α]25 D +108 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 235 (4.6), 254 (4.51), 292 (4.03), 494 (4.32), 528 (4.14), 589 (2.74) nm; 1H and 13C NMR spectroscopic data, Table 1; (+)-HRESIMS m/z 761.3019 [M + H]+ (calcd for C38H49O16, 761.3015); (+)-HRESIMS m/z 783.2838 [M + Na]+ (calcd for C38H48NaO16, 783.2835). 2-Deoxy-L-fucose-L-rhodinose-2-deoxy-L-fucose-β-rhodomycinone (7): red powder; [α]25 D −230 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 235 (4.4), 295 (3.73), 497 (3.84), 513 (3.83), 531 (3.84), 587 (3.76) nm; 1H and 13C NMR spectroscopic data, Table 2; (−)-HRESIMS m/z 759.2881 [M − H]− (calcd for C38H47O16, 759.2870). L-Rhodinose-2-deoxy-L-fucose-2-deoxy-L-fucose-10-decarbomethoxy-ε-rhodomycinone (8): red powder; [α]D25 −186 (c 0.03, CDCl3); UV (MeOH) λmax (log ε) 240 (4.84), 300 (3.93), 497 (4.04), 520 (3.9), 533 (3.91) nm; 1H and 13C NMR spectroscopic data, Table 2; (+)-HRESIMS m/z 767.2877 [M + Na]+ (calcd for C38H48NaO15, 767.2885). 2-Deoxy-L-fucose-2-deoxy-L-fucose-β-rhodomycinone (9): red powder; [α]25 D −102 (c 0.016, MeOH); UV (MeOH) λmax (log ε) 236 (4.89), 253 (4.83), 292 (4.37), 494 (4.64), 528 (4.48), 588 (3.52) nm; 1H and 13C NMR spectroscopic data, Table 2; (−)-HRESIMS m/ z 645.2185 [M − H] − (calcd for C 32 H 37 O 14 , 645.2189); (+)-HRESIMS m/z 669.2052 [M + Na]+ (calcd for C32H38NaO14, 669.2154). 2-Deoxy-L-fucose-2-deoxy-L-fucose-2-deoxy-L-fucose-β-rhodomycinone (10): red powder; [α]25 D +63 (c 0.1, CDCl3−DMSO, 10:1); UV (MeOH) λmax (log ε) 252 (4.09), 296 (3.56), 499 (3.76), 521 (3.62), 534 (3.6) nm; 1H and 13C NMR spectroscopic data, Table 2; (−)-HRESIMS m/z 775.2824 [M − H]− (calcd for C38H47O17, 775.2819). L -Cinerulose-2-deoxy- L -fucose-2-deoxy- L -fucose-β-rhodomycinone (11): red powder; [α]25 D +28 (c 0.12, CDCl3); UV (MeOH) λmax (log ε) 240 (4.31), 296 (3.56), 497 (3.73), 533 (3.55) nm; 1H and 13C NMR spectroscopic data, Table 2; (−)-HRESIMS m/z 757.2715 [M − H]− (calcd for C38H45NO16, 757.2713); (+)-HRESIMS m/z 781.2679 [M + Na]+ (calcd for C38H46NaO16, 781.2678). L -Cinerulose-2-deoxy-L -fucose- L-rhodinose-β-rhodomycinone (12): red powder; [α]25 D +37 (c 0.06, CDCl3−DMSO 10:1); UV (MeOH) λmax (log ε) 256 (4.02), 293 (3.49), 520 (3.68), 499 (3.51), 534 (3.02) nm; 1H and 13C NMR spectroscopic data, Table 2; (+)-HRESIMS 765.2715 [M + Na]+ (calcd for C38H46NaO15, 765.2729). Cytotoxicity Assay. Standard MTT assays employing MDA-MB435, NCI-H460, MDA-MB-231, HCT-116, HepG2, and MCF10A cell

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00212. Spectra of 1D and 2D NMR for compounds 1−12 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (J. Ju): +86-20-89023028. E-mail: [email protected]. ORCID

Jianhua Ju: 0000-0001-7712-8027 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the National Natural Science Foundation of China (81425022, U1501223, and 41476133), the Program of Chinese Academy of Sciences (XDA11030403), Natural Science Foundation of Guangdong Province (2016A030312014), and the Syngenta Ph.D. Fellowship awarded to C.G. We thank the analytical facility at SCSIO for recording spectroscopic data.

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REFERENCES

(1) Olano, C.; Méndez, C.; Salas, J. A. Nat. Prod. Rep. 2009, 26, 628− 660. (2) Niemi, J.; Ylihonko, K.; Hakala, J.; Pärssinen, R.; Kopio, A.; Mäntsälä, P. Microbiology 1994, 140, 1351−1358. (3) Jarmo, N.; Mikko, M.-K.; Gunter, S.; Pekka, M. Top Curr. Chem. 2008, 282, 75−99. (4) McGowan, J. V.; Chung, R.; Maulik, A.; Piotrowska, I.; Walker, J. M.; Yellon, D. M. Cardiovasc. Drugs Ther. 2017, 31, 63−75. (5) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Pharmacol. Rev. 2004, 56, 185−229. (6) Rooverts, D. J.; Van Vliet, M.; Bloem, A. C.; Lokhorst, H. M. Leuk. Res. 1999, 6, 539−548. 1288



Article

(7) Duan, C.; Yao, Y.; Wang, Z.; Tian, X.; Zhang, S.; Zhang, C.; Ju, J. Chin. J. Mar. Drugs 2010, 29, 12−20. (8) Mo, X.; Wang, Z.; Wang, B.; Ma, J.; Huang, H.; Tian, X.; Zhang, S.; Zhang, C.; Ju, J. Biochem. Biophys. Res. Commun. 2011, 18, 341− 347. (9) Mo, X.; Huang, H.; Ma, J.; Wang, Z.; Wang, B.; Zhang, S.; Zhang, C.; Ju, J. Org. Lett. 2011, 13, 2212−2215. (10) Mo, X.; Ma, J.; Huang, H.; Wang, B.; Song, Y.; Zhang, S.; Zhang, C.; Ju, J. J. Am. Chem. Soc. 2012, 134, 2844−2847. (11) Gui, C.; Li, Q.; Mo, X.; Qin, X.; Ma, J.; Ju, J. Org. Lett. 2015, 17, 628−631. (12) Gui, C.; Mo, X.; Gu, Y.-C.; Ju, J. Org. Lett. 2017, 19, 5617− 5620. (13) Bonfante, V.; Bonadonna, G.; Villani, F.; Martini, A. Recent Results Cancer Res. 1980, 74, 192−199. (14) Olano, C.; Gómez, C.; Pérez, M.; Palomino, M.; Pineda-Lucena, A.; Carbajo, R. J.; Braña, A. F.; Méndez, C.; Salas, J. A. Chem. Biol. 2009, 16, 1031−1044. (15) Zhang, Y.; Huang, H.; Chen, Q.; Luo, M.; Sun, A.; Song, Y.; Ma, J.; Ju, J. Org. Lett. 2013, 15, 3254−3257. (16) Reddy, G. C.; Sahai, R.; Fehlhaber, H. W.; Ganguli, B. N. J. Antibiot. 1985, 38, 1423−1425. (17) Ding, B.; Yuan, J.; Huang, X.; Wen, W.; Zhu, X.; Liu, Y.; Li, H.; Lu, Y.; He, L.; Tan, H.; She, Z. Mar. Drugs 2013, 11, 4961−4972.

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Cytotoxic Anthracycline Metabolites from a Recombinant

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