Amycolamycins A and B, Two Enediyne-Derived Compounds from a

Letter Cite This: Org. Lett. 2017, 19, 6208-6211

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Amycolamycins A and B, Two Enediyne-Derived Compounds from a Locust-Associated Actinomycete Shi Ying Ma,†,∥ Yong Sheng Xiao,†,∥ Bo Zhang,†,∥ Fen Li Shao,† Zhi Kai Guo,§ Juan Juan Zhang,† Rui Hua Jiao,† Yang Sun,† Qiang Xu,† Ren Xiang Tan,*,†,‡ and Hui Ming Ge*,† †

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China § Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Hainan 571101, China ‡

S Supporting Information *

ABSTRACT: Two novel enediyne-derived natural products, amycolamycins A and B (1 and 2), were characterized from a locust-associated actinomycete Amycolatopsis sp. HCa4. Amycolamycins A and B contain a unique 2-(cyclopenta[a]inden-5-yl)oxirane core with suspected enediyne polyketide biosynthetic origin. Sequencing and analysis of the acm biosynthetic gene cluster allowed us to propose the biosynthetic pathway of 1 and 2. Moreover, amycolamycin A (1) was selectively cytotoxic to the M231 breast cancer cell line.

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(2), with 1 showing selective cytotoxicity to M231 breast cancer cell line (Scheme 1). Sequencing and analysis of the acm biosynthetic gene cluster allowed us to propose a convergent pathway for the assembly of amycolamycins. Amycolamycin A (1) was isolated as a pale yellow solid. The molecular formula of 1 was determined to be C39H40ClNO14 ([M + Na]+, m/z 804.2038, calcd for [C39H4035ClNO14Na]+, 804.2035), indicating 20 degrees of unsaturation. Analyses of the 1 H, 13C, HSQC NMR spectra of 1 allowed the assignment of all

nediyne natural products represent one of the most fascinating families of secondary metabolites, owing to their unusual molecular architecture and potent DNA damaging activity.1 They are rare in nature but highly druggable in terms of hit rate. From the 12 natural enediynes characterized so far (Figure S1),2 twoneocarzinostatin and calicheamicinhave been developed and approved as clinical anticancer drugs in Japan and the United States, respectively, and at least four (C1027, uncialamycin, dynemicin, and esperamicin) are in varying stages of drug development.3 In stark contrast with the limited number of enediynes discovered to date, the gene clusters for enediyne biosynthesis are widely present in actinomycetes.2 One likely reason for this contradiction is that the enediyne cores are notorious labile and easily undergo the Bergman or Myers−Saito rearrangement to afford a transient benzenoid diradical, leading to final degradation and failed isolation.1 However, three enediyne-derived natural products including cyanosporasides, fijiolides, and sporolides (Figure S1) were isolated as cycloaromatized form from four actinomycetes.4 These enediynederived compounds have drawn substantial interest because the structural stability provided outstanding opportunities to decipher the genetic and biochemical basis for enediyne biosynthetic machinery and offered additional access to redesigning these molecules through bioengineering and chemical synthesis.4b,5 In our continuing effort to discover novel/bioactive natural products from microorganisms derived from insects,6 we set out to explore metabolites from a locust-associated actinomycete Amycolatopsis sp. HCa4. This led to the discovery of two unusual stable enediye-derived compounds, amycolamycins A (1) and B © 2017 American Chemical Society

Scheme 1. Structures of Amycolamycins A (1) and B (2)

Received: October 9, 2017 Published: November 1, 2017 6208

DOI: 10.1021/acs.orglett.7b03113 Org. Lett. 2017, 19, 6208−6211

Letter

Organic Letters protons to their corresponding carbon atoms, leaving six exchangeable protons (Table S1). 2D NMR correlations, including HSQC, HMBC, and 1H−1H COSY experiments (Figure 1), showed four subunits consisting

Figure 2. ΔδS−R value in ppm for S- and R-bis-MTPA-esters for amycolamycins A (1) and B (2).

indicated all stereocenters of 2 had the same relative configurations with those in 1 except for C-31, since no obvious NOE correlation was observed between H-31 and H-37 in 2 (Figure S2). This assignment was further supported by modified Mosher’s ester method. As shown in Figure 2, the ΔδS−R value pattern around C-31 in 2 is opposite to that in 1, permitting the assignment of the 31S of 2, while absolute configurations for other chiral carbons remain identical to 1. The cyclopenta[a]indene carbon skeleton of amycolamycins A and B is structurally similar to the cycloaromatized product of natural enediyne antitumor antibiotics such as neocarzinostatin, maduropeptin, or C-1027,1b raising the intriguing question if compounds 1 and 2 are also derived from enediyne precursors through a Bergman cycloaddition followed by quenching with two hydrogens (Figure 3). To explore the molecular basis for the biosynthesis of amycolamycins A and B, the genome of Amycolatopsis sp. HCa4 was queried for the presence of enediyne polyketide synthase containing genes.6a Only one enediyne gene cluster (acm) was identified (accession no. MF990204), sharing high homologue with spo (sporolide gene cluster) and mdp (maduropeptin gene cluster). The acm gene cluster spans a ∼76 kb contiguous DNA region consisting of minimally 65 genes responsible for biosynthesis, regulation, and resistance (Figure 3A). Bioinformatics analysis allowed us to propose a convergent assembly pathway for biosynthesis of amycolamycins A and B. Four genes products, AcmA2 to AcmA5, have the closest sequence homology with enzymes involved in aminosugar biosynthesis of maduropeptin from Actinomadura madurae ATCC39144, with sequence identities ranging from 59% to 74% (Table S3).9 Thus, AcmA2 to AcmA5 is responsible for catalyzing four successive steps starting from NDP-glucose to NDP activated aminosugar building block in amycolamycin (Figure 3B). Three genes including acmB, acmB1, and acmB2 encode proteins similar to MdpB, MdpB1, and MdpB2, which are responsible for biosynthesis of 3,6-dimethylsalicylyl-CoA in maduropeptin (Table S3).9 AcmB consists of characteristic domains for iterative type-I PKS catalyzing the formation of 6methylsalicylic acid, which is then activated by AcmB2, a CoA ligase, to form 6-methylsalicylic CoA. The subsequent Cmethylation step was catalyzed by AcmB1, affording 3,6dimethylsalicylyl CoA (Figure 3B).10 Ten genes (acmP1−acmP10) were identified to biosynthesize 2-chloro-3-hydroxy-4,5-dimethoxymandelate moiety (Table S3). AcmP1 has moderate sequence homologue (57% identity) with

Figure 1. Key 2D NMR correlations for compound 1.

of a 2-hydroxy-3,6-dimethylbenzamide, an aminosugar, a 2chloro-3-hydroxy-4,5-dimethoxymandelate, and 2-(cyclopenta[a]inden-5-yl)oxirane core (for a detailed elucidation see the Supporting Information). The diagnostic HMBC correlations of H-1/C-30, H-7/C-34, H-15/C-8, and H-18/C-21 connected the above four units and established the complete planar structure of amycolamycin A (1) as shown in Figure 1. The relative configuration of 1 was determined by interpretation of its NOESY spectrum (Figure 1) and coupling constants. The magnitude of the 1JCH coupling constant for anomeric proton (H-15, 161 Hz) established the β-configuration of this center,7 while a large diaxial coupling constant (7.8 Hz) between H-15 and H-16 indicated an axial configuration for H16. The strong NOE correlations of H-16 with H3-20 and H-18 revealed that Me-20 and H-18 are in the equatorial and axial positions, respectively. NOE correlation of H3-38 with H-9 indicated the bridging system was stacked on the cyclopenta[a]indene moiety and established the relative configuration of C9. The observed NOE interaction between H-7 and the anomeric proton H-15 indicated the trans-orientation of the C-8 glycoside and C-7 ether bond. Moreover, diagnostic NOE correlations from H-2 to H-14 and from H-31 to H-37 established the relative configurations of C-2 and C-31, respectively. The small vicinal coupling constant (2.1 Hz) between H-1 and H-2 revealed the geometry of the epoxy ring was trans as depicted in Figure 1.8 The absolute configurations of 1 were determined by the modified Mosher’s method. Treatment of 1 with R- and S-MTPA chloride in anhydrous pyridine solution yielded the corresponding S-(1a) and R-bis-MTPA-ester (1b) (Figure 2). The proton chemical shifts (Table S2) of them were assigned by analysis of 1 H NMR, COSY, and HSQC spectral data. Thus, the absolute configurations of amycolamycin A aglycon were assigned as 1R,2R,7R,8S,9R,31R on the basis of the pattern of ΔδS−R values (Figure 2). Amycolamycin B (2), a pale yellow amorphous solid, also had the molecular formula C39H40ClNO14 on the basis of HRESIMS data ([M + Na]+, m/z 804.2026, calcd for [C39H4035ClNO14Na]+, 804.2035). The 1H and 13C NMR data (Table S1) resembled those of 1. Explanation of the twodimensional COSY, HSQC, and HMBC NMR spectra revealed that 2 possessed the same planar structure with 1 (Figure S2). Analyses of its NOESY (Figure S2) and coupling constant data 6209

DOI: 10.1021/acs.orglett.7b03113 Org. Lett. 2017, 19, 6208−6211

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Figure 3. Biosynthesis of amycolamycins A and B. (A) acm biosynthetic gene cluster. (b) Proposed biosynthetic pathway for amycolamycins A and B: (i) aminosugar, (ii) 3,6-dimethylsalicylyl-CoA, (iii) 2-chloro-3-hydroxy-4,5-dimethoxymandelate, (iv) enediyne core, assembly of four units, and Bergman cyclization leading to form amycolamycins A and B.

clusters of cycloaromatized enediyne compounds such as sporolides and cyanosporasides (Figure S1).4b,5a It is noteworthy that a single gene, acmA, was indeed identified within the acm gene cluster, whose gene product was a homologue to CagA (35% identity) and NcsA (36% identity),12 the apoproteins for C-1027, and neocarzinostatin, respectively. However, exhaustive attempts to detect activated enediyne chromophores by LC−MS or to test the DNA cleavage activity using biochemical induction assay (BIA)13 in its crude extract or fermentation broth proved unsuccessful. Amycolamycins A and B (1 and 2) were tested on a panel of cancer cell lines. Intriguingly, compound 1 showed cytotoxicity only against M231 cell line with its IC50 of 7.9 μM. However, no obvious activities were observed in HL60, A549, MCF7, and BL6F10 cell lines (Figure 4A). In contrast, compound 2 is not active on tested cells (Figure S3) including the M231 cell line (Figure 4B), clearly signifying the importance of the stereochemistry at C-31. To further understand the underlying mechanism of cytotoxicity of 1, induction of apoptosis was investigated by flow cytometry. Compound 1 was shown to induce apoptosis in a dose-dependent manner in M231 cells (Figure 4C). Furthermore, Western blot results indicated that compound 1 significantly increased the generation of cleaved caspase-3 (Figure 4D), which was a central process of cell apoptosis. These data suggested that amycolamycin A (1) suppressed the proliferation of M231 cells by inducing apoptosis through activation of caspase-3. In conclusion, we present here the isolation and characterization of two novel enediyne-derived natural products, amycolamycins A (1) and B (2), from A. sp. Hca4. Identification and analysis of the acm biosynthetic gene cluster suggested amycolamycins are formed from the convergence of four building blocks, which set the stage for future exploitation of

SpoT1, a biochemically characterized dioxygenase in sporolide biosynthesis from Salinispora tropica.5a Therefore, the initial step could be catalyzed by AcmP1 converting p-hydroxyphenylpyruvate (p-HPPA) to p-hydroxymandelate (p-HMA), which was then activated and tethered on AcmP2, an A-PCP (adenylationpeptidyl carrier protein) didomain protein. The following steps including hydroxylation, chlorination, and O-methylation are proposed to be occurred on an AcmP2-tethered substrate by a phenol hydroxylase (AcmP3), a monooxygenase (AcmP9), a methyltransferase (AcmP6), and a halogenase (AcmP4) (Figure 3B). However, the precise timing of these steps remains unclear. The hallmark of the enediyne family compounds is the enediyne core moiety, which is biosynthesized by an iterative type I PKS. AcmE, showing high sequence identity to other known enediyne PKSs, is envisioned to synthesize a polyketide backbone11 that will be further processed by accessory enzymes including AcmE2 to AcmE11, AcmD2, AcmL and AcmM,12 to finally form enediyne core (Figure 3B). The assembly of amycolamycin features a convergent pathway that employs varying coupling strategy. The aminosugar is attached to enediyne core catalyzed by AcmA6 glycotransferase, the coupling between 3,6-dimethylsalicylic CoA and aminosugar is likely catalyzed by AcmB3, an acetyltransferase; and 2-chloro-3hydroxy-4,5-dimethoxymandelate moiety was condensed to enediyne core by a type II condensation enzyme, AcmP10. The enzyme responsible for the formation of ether bridge between C-7 and C-34 is still enigmatic. Nine-membered enediynes like C-1027, neocarzinostatin, kedarcidin, and maduropeptin (Figure S1) are isolated as a complex consisting of a labile chromophore and an apoprotein in 1:1 ratio, the latter of which functions as a stabilizer and is encoded by a small apoprotein gene within the respective gene cluster.1 In contrast, no such genes were found in the gene 6210

DOI: 10.1021/acs.orglett.7b03113 Org. Lett. 2017, 19, 6208−6211

Organic Letters

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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (81522042, 21572100, 81421091, 81500059, 81673333, 21672101, and 21661140001).

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Figure 4. Compound 1 induces apoptosis through activation of caspase3 in M231 cells. (A) HL60, A549, MCF7, BL6F10, and M231 cell lines were treated with 10 μM compound 1 for 72 h, and cell viability was determined by MTT assay. (B) Varied concentration of compounds 1 or 2 was added to the medium of M231 cells, respectively, and cell viability was measured by MTT assay after 72 h. DMSO was used as vehicle control. (C) Apoptosis of M231 cells treated with varied concentration of compound 1 analyzed by flow cytometry and using DMSO as vehicle control (annexin V-FITC+PI− cells in the lower-right quadrant are early apoptotic, and the annexin V-FITC+PI+ cells in the upper-right quadrant are late-apoptotic). (D) Western blot analysis of caspase-3 and cleaved caspase-3 levels in M231 cells treated with compound 1 for 24 h and βactin used as loading control.

bioengineering and biosynthetic study. Genome mining in the NCBI and JGI database led to identification of one almost identical homologous gene cluster in Amycolatopsis rifamycinica DSM44544 (Figure S4, Table S4) and three highly similar ones in Xiangella phaseoli CGMCC4.7038, Micromonospora narathiwatensis DSM45248, and M. eburnea DSM44814 (Figure S4, Tables S5−7), which may facilitate the discovery of active enediynes in the near future.

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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.orglett.7b03113. Experimental details, spectroscopic data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ren Xiang Tan: 0000-0001-6532-6261 Hui Ming Ge: 0000-0002-0468-808X Author Contributions ∥

S.Y.M., Y.S.X., and B.Z. contributed equally.

Notes

The authors declare no competing financial interest. 6211

DOI: 10.1021/acs.orglett.7b03113 Org. Lett. 2017, 19, 6208−6211