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actinomycetes derived from insects are considered to be a promising reservoir of novel natural products. Mud dauber wasps are solitary Hymenoptera ins...
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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Strepantibins A−C: Hexokinase II Inhibitors from a Mud Dauber Wasp Associated Streptomyces sp. Ya-Jie Song,† Hong-Bo Zheng,‡ Ai-Hong Peng,† Jia-Hui Ma,† Dan-Dan Lu,† Xia Li,†,§ Hang-Yu Zhang,# and Wei-Dong Xie*,†,§ †

Department of Pharmacy, College of Marine Science, Shandong University at Weihai, Weihai 264209, People’s Republic of China Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Science, Shandong University, Jinan 250012, People’s Republic of China § Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, People’s Republic of China # Department of Biomedical Engineering, Faculty of Electronic Information and Electrical Engineering, Dalian University of Technology, Dalian 116024, People’s Republic of China

J. Nat. Prod. Downloaded from pubs.acs.org by AUBURN UNIV on 04/23/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Two new p-terphenyls, strepantibins A and B (1 and 2), along with the first representative of a naturally occurring bisphenyltropone, strepantibin C (3), were characterized from a Streptomyces sp. associated with the larvae of the mud dauber wasp Sceliphron madraspatanum. Their structures were determined by high-resolution electrospray ionization mass spectrometry, NMR, and X-ray crystallography data interpretation. Strepantibins A−C inhibited hexokinase II (HK2) activity and displayed antiproliferative activity against hepatoma carcinoma cells HepG-2, SMMC-7721 and plcprf-5. In SMMC-7721 cells treated with strepantibin A, the morphological characteristics of apoptosis were observed.

I

In the course of cancer cells’ metabolism, HK2 catalyzes the rate-limiting and first obligatory step of glucose metabolism, when glucose is transformed into glucose-6-phosphate.13,14 Hence, HK2 is an essential factor in cancer initiation and may help drive the tumor cells’ growth. It stands as a promising therapeutic target, although no HK2 inhibitors have been developed clinically. With the aim of discovering new HK2 inhibitory compounds from actinomyces, we isolated actinomycetes from the mud dauber wasp, Sceliphron madraspatanum, and its nest. A Streptomyces sp. designated as N1510.2 was obtained from a larva removed from one intact cocoon. The 16S rRNA nucleotide sequence (accession number MH211598) is similar to those of S. griseoruber (GenBank FJ919750) and S. antibioticus (GenBank AB184184), displaying a 98.1 and 98.0% similarity, respectively (Figure S2). In preliminary screening, the ethyl acetate extracts of a culture of the N1510.2 strain showed HK2 inhibitory activity. Bioactivity-guided isolation from extracts of large-scale cultures led to the identification of strepantibins A−C (1−3).

nsects, especially those dwelling in soil, sometimes recruit microorganisms from their surroundings to form complex symbiotic associations.1,2 Investigations on symbiotic associations between actinomycetes and insects such as leaf-cutter ants,3 fungus-growing termites,4 European beewolf Philanthus triangulum,5 and southern pine beetle Dendroctonus f rontalis6 have revealed that actinomycetes can provide health benefits for insect larvae through their food by producing antimicrobial compounds. Typically, symbiotic actinomycetes exist in the hosts’ integument, guts, or specialized mycangiums, where they exchange small molecules with their hosts.7 These unusual microhabitats, which are distinctly different from those of soil microorganisms, promote the actinomycetes associated with insects to produce secondary metabolites with biological activity and unusual chemical scaffolds, e.g., dentigerumycin and selvamicin from leaf-cutter ants system.8,9 Accordingly, actinomycetes derived from insects are considered to be a promising reservoir of novel natural products. Mud dauber wasps are solitary Hymenoptera insects. They construct nests with humid soil or sand mixed with their oral secretions for reinforcement. In the course of nest building and food storage, a variety of soil actinomycetes will be incorporated into their nests.10,11 Hexokinase II (HK2) is rarely expressed in normal tissues. On the contrary, it is especially overexpressed in cancer cells.12 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 3, 2018

A

DOI: 10.1021/acs.jnatprod.8b00821 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 1. 1H and

13

Article

C NMR (DEPT) Data of 1−3a 1

no.

δC mult.

1 2 3 4 5 6 7

199.7 s 115.3 s 165.5 s 117.4 d 154.3 s 76.0 s 54.9 t

8 9 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ MeO

205.3 s 31.1 q 133.9 s 131.8 d 128.1 d 127.5 d 128.1 d 131.8 d 138.9 s 129.4 d 129.0 d 129.6 d 129.0 d 129.4 d 57.4 d

2 δH (J in Hz)

δC mult.

6.81, s

186.2 112.1 160.3 156.8 139.6 134.5 118.2

2.92, d (15.2) 3.33, d (15.2) 1.92, s 7.34,b m 7.32,b m 7.24, m 7.32,b m 7.34,b m 7.78, m 7.43,b m 7.42,b m 7.43,b m 7.78, m 3.97, s

3 δH (J in Hz)

s s s d s s s

6.82, s

25.7 q 25.7 q 132.1 s 130.9 d 128.5 d 128.2 d 128.5 d 130.9 d 134.5 s 129.7 d 129.3 d 130.6 d 129.3 d 129.7 d

1.76, s 1.76, s 7.65, 7.41, 7.32, 7.41, 7.65,

brd (7.3) t (7.3) brt (7.3) t (7.3) brd (7.3)

7.92, m 7.52,b m 7.52,b m 7.52,b m 7.92, m

δC mult. 176.0 124.3 162.9 120.7 150.3 191.1 118.4

s s s d s s s

203.6 s 28.5 q 136.1 s 131.2 d 128.5 d 127.8 d 128.5 d 131.2 d 137.9 s 129.5 d 129.0 d 130.4 d 129.5 d 129.3 d 58.3 d

δH (J in Hz)

7.30,b s

2.37, s 7.25, brd (7.3) 7.38, t (7.3) 7.31,b m 7.38, t (7.3) 7.25, brd (7.3) 7.79, m 7.46,b m 7.46,b m 7.46,b m 7.79, m 3.86, s

Assignments were based on 1H−1H COSY, gHMQC, and HMBC experiments in acetone-d6 [δH (500 MHz) and δC (125 MHz) in ppm, multiplicity (J in Hz), TMS as internal standard]. bOverlapping signals.

a

Figure 1. Key 1H−1H COSY (black bold lines), HMBC (blue arrows), and NOESY (red arrows) correlations of 1 and its ORTEP plot of crystal structure.

unsaturation constitute a cyclohexadienone substructure (Figure 1). In the HMBC spectrum, the correlations of H-4 to C-2, C-3, C-6 and C-1″, H-2′ to C-2, and H-2″ to C-5 further confirmed the presence of cyclohexadienone moiety and revealed the locations of two phenyls at C-2 and C-5 of the middle ring. The HMBC correlation between methoxyl protons and C-3, and the NOESY correlation between methoxyl protons and H-4, indicated the methoxyl group is located at C-3. The positions of the acetonyl and hydroxy groups can be deduced by the HMBC correlations of methylene protons to carbons at δC 205.3 (C-8), δC 154.3 (C-5), and δC 76.0 (C-6). The skeleton of 1 and the R absolute configuration with Flack parameter 0.04(5) of C-6 were finally determined by X-ray diffraction (Cu Kα) (Figure 1). Strepantibin B (2) has a molecular formula C21H17NO2 determined by the quasi-molecular ion in positive HRESIMS. The aromatic signals in 1H NMR of 2 are very similar to those of 1, indicating the presence of a p-terphenyl motif with two monosubstituted phenyls (Table 1). The structure of the middle ring and the positions of two phenyls were supported by the HMBC correlations (Figure 2). In addition to aromatic proton signals in the 1H NMR, there is a singlet of two methyl groups at δH 1.76 ppm, whose associated protons showed only

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RESULTS AND DISCUSSION Strepantibin A (1) was isolated as a yellowish solid. Its molecular formula was deduced to be C22H20O4 by positive high-resolution electrospray ionization mass spectrometry (HRESIMS), indicating 13 degrees of unsaturation. The 1H NMR spectrum showed the signals of 11 aromatic protons, the signal of methoxyl at δH 3.97 ppm, methyl at δH 1.92 ppm, and two of AB coupling system of methylene at δH 3.33 (d, J = 15.2 Hz) and 2.92 (d, J = 15.2 Hz) ppm (Table 1). Ten coupling aromatic proton signals from δH 7.30 to 7.80 ppm and four pairs of overlapping carbon signals at δC 128.1, 129.0, 129.4, and 131.8 ppm indicated the presence of two monosubstituted phenyl groups, which was further confirmed by 1H−1H COSY correlations (Figure 1). The chemical shifts of methyl and methylene protons, along with the HMBC correlations of methyl protons with carbonyl carbon at δC 205.3 (C-8) and methylene carbon at δC 54.9 (C-7), supported an acetonyl group.15 The remaining six carbons along with four degrees of B

DOI: 10.1021/acs.jnatprod.8b00821 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Biosynthesis Pathway. Both 1 and 2, possessing a sixmembered middle ring, are p-terphenyl derivatives possibly derived from cinnamic acids.18 The p-terphenyl 4-methoxy-3,6diphenyl-3,5-cyclohexadiene-1,2-dione (4) was initially identified from a Phoma species19 and could be a biosynthetic precursor of 1. Acetoacetyl-CoA, obtained by a Claisen condensation reaction between acetyl-CoA and malonyl-CoA, could react with 4 by nucleophilic addition followed by hydrolysis and decarboxylation to give 1 (Scheme S1). Previously, two p-terphenyls with a thiazole condensed with the middle aromatic ring have been identified from a marine Streptomyces.20 It is rare that there is an oxazole in strepantibin B (2). Considering the propylidene group is often the result of an acetone addition, 2 is probably produced in the isolation process. Tropolonoids, including tropones and tropolones, are nonbenzenoid aromatic compounds possessing a core of 2,4,6cycloheptatrien-1-one. Many natural products such as nezukone, hinokitiol, colchicines, and stipitatic acid contain a tropolone or tropone skeleton.21 Strepantibin C (3) is, to our knowledge, the first naturally occurring tropone derivative with two phenyl substituents. Although the precursors and biosynthetic gene cluster of 3 remain to be explored by biosynthetic experiments, 3 is most likely derived from 1 by an intramolecular Aldol reaction and dehydrogenation reaction (Scheme S1).22 Bioactivity Assays. Hexokinase II (HK2), a rate-limiting enzyme of glycolysis, participates in tumor glycolysis and the progression of various cancers and has been used as a target in cancer drug research in recent years.23 Metformin, 2deoxyglucose, and 3-bromopyruvate are the common HK2 inhibitors. Recently, benserazide was identified as a selective HK2 inhibitor with an IC50 value 5.52 μM with a better binding affinity and efficacy than other reported HK2 inhibitors, thus representing the most potent HK2 inhibitor.24 Compounds 1−3 exhibited in vitro HK2 inhibitory activity with IC50 values of 9.8, 34.6, and 31.2 μM, respectively (Figure 4). To determine if strepantibins inhibited competitively to

Figure 2. Key 1H−1H COSY (black bold lines) and HMBC (blue arrows) correlations of 2 and its ORTEP plot of crystal structure.

HMBC correlations to one carbon at δC 118.2 ppm. This suggested the presence of an isopropyl group. The chemical shift of carbon at δC 118.2 ppm is the result of its connections to N and O, simultaneously, to form an oxazole with pterphenyl. The presence of the oxazole and the skeleton of 2 were finally confirmed by X-ray diffraction (Mo Kα) (Figure 2). Strepantibin C (3) has a molecular formula of C22H18O4, as determined on the basis of the quasi-molecular ion in positive HRESIMS and negative HRESIMS. Its 1H NMR displayed 11 aromatic proton signals similar to those of 1, although one aromatic proton signal was shifted downfield to δH 7.30 ppm overlapping with the signals of the phenyl protons (Table 1). The proton signal at δH 3.86 ppm and the corresponding carbon signal at δC 58.3 ppm were assigned to a methoxyl group. An acetyl group was identified from the signals of methyl protons at δH 2.38 ppm and corresponding carbon at δC 28.5 ppm, and the observed HMBC correlation between methyl protons and carbonyl carbon at δC 203.6 ppm. Six aromatic carbon signals at δC 176.0, 162.9, 150.3, 124.3, 120.7, and 118.4 ppm, along with the carbonyl carbon signal at δC 191.1 ppm, supported the presence of a cycloheptatrienone moiety, namely, tropone or tropolone.16,17 The substructures of two monosubstituted phenyls and tropone moiety, and the positions of methoxyl, hydroxy, and acetyl groups were established by key 1H−1H COSY and HMBC correlations (Figure 3). The 1H−1H COSY correlations between δH 7.25

Figure 3. Key 1H−1H COSY (black bold lines) and HMBC (blue arrows) correlations of 3 and its ORTEP plot of crystal structure.

(H-2′), 7.38 (H-3′), and 7.31 (H-4′) ppm and 1H−1H COSY correlations between δH 7.79 (H-2″) and 7.46 (H-3″ and 4″) ppm supported the presence of two monosubstituted phenyl groups. In the HMBC spectrum, the correlations of δH 7.30 (H-4) to δC 124.3 (C-2), 162.9 (C-3), 150.3 (C-5), and 191.1 (C-6) identified the signals of these four carbons. The HMBC correlations δH 7.25 (H-2′)/δC 124.3 (C-2), δH 7.79 (H-2″)/ δC 150.3 (C-5) and δH 3.86 (OCH3)/δC 162.9 (C-3) supported the positions of two phenyl groups and the methoxy group. The methyl protons showed HMBC correlations with δC 118.4 (C-7) and 203.6 (C-8) ppm. Finally, the structure of 3 including the tropone was confirmed by X-ray diffraction (Mo Kα) (Figure 3). Hence, the downfield shift of H-4 signal to δH 7.30 ppm is mostly the result of partial positive charge of the tropone as it tries to attain the six π electron state required for aromaticity.

Figure 4. Dose−response inhibition of HK2 activity by strepantibitins A−C (1−3). Data are expressed as the mean ± SD of triplicate experiments.

HK2, we performed Lineweaver−Burk plots experiments and measured Michaelis−Menten constants (Km). The Lineweaver−Burk plots changed with treatment of the inhibitors. Our findings showed that the Km of strepantibins were A-0.43 mM, B-0.29 mM, and C-0.31 mM and 0.21 mM for the control group (HK2 without treatment), where the slope of line increased with the same intercept. These results indicated that strepantibins inhibited HK2 in a competitive and efficient way C

DOI: 10.1021/acs.jnatprod.8b00821 J. Nat. Prod. XXXX, XXX, XXX−XXX

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measured on Agilent 8453E UV−visible spectroscopy system. IR spectra were obtained from a Nicolet NEXUS 470 FT-IR spectrometer in KBr plate. 1H, 13C NMR (DEPT), and 2D NMR spectra were recorded on Bruker AVANCE 500 spectrometer. Chemical shifts are given on the δ (ppm) scale with TMS as internal reference. HPLC-HRESIMS were acquired on Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS. Preparative HPLC isolation were carried on Agilent 1260 Infinity II (YMC Triart C18, 20 × 250 mm). Column chromatography (CC) was carried out on silica gel (200−300 and 300−400 mesh) and TLC on silica gel (GF254 10−40 μm) with both materials supplied by Qingdao Marine Chemical Factory in China. Actinomyces Strains. A nest of the mud dauber wasp S. madraspatanum was collected in suburbs of Dezhou, Shandong province, China in October 2015. The whole nest chambers were split with sterile scalpel, and the cocoons removed from the nest chambers, and a larva taken out from an intact cocoon. After washing with sterilized water and 95% ethanol for 2 min, the larva body was ground in 2 mL of sterile water. Suspensions (200 μL) were spread on three Petri dishes containing Gause’s synthetic medium (GAU; 20 g/L amylogen, 1 g/L KNO3, 0.5 g/L NaCl, 0.5 g/L K2HPO4·H2O, 0.5 g/L MgSO4·H2O, and 0.01 g/L FeSO4·H2O) supplemented with potassium dichromate (100 μg/mL) and nalidixic acid (20 μg/mL) as antifungal and antibacterial agents. The dishes were cultured for 2 weeks at 28 °C. The actinomycetes colonies were isolated and subcultured repeatedly at the same conditions until pure morphologies were obtained (Figure S2). The pure culture was stored in 25% glycerol of water solution at −20 °C to further analyses. PCR amplification of the 16S rRNA (rRNA) from actinomyces was performed separately using 2 primers Fd2 (5′-GAGTTTGATCATGGCTCAG-3′) and 16Sr (5′-TTGCGGGACTTAACCCAACA-3′) and sequenced by Shanghai Sangon Biotech Co., China. PCR mixture (50 μL) consists of 1× 10× PCR buffer, 0.2 mmol/L d NTP mix, 0.1−1 μmol/L primer, 1 U pfu DNA polymerase, 10 pg-1 μg/L emplate DNA, 10% DMSO, and sterilized deionized water. The amplification procedure includes preheating at 94 °C for 10 min, consecutive incubation 31 cycles at 94 °C for 45 s, 52 °C for 45 s, and 72 °C for 105 s, and a postincubation at 72 °C for 5 min. PCR products were purified using PCR Purification Kit (Shanghai Sangon Biotech Co., China). Positive bands on a 1.5% agarose gel were directsequenced at the Shanghai Sangon Biotech Co., China. The nucleotide sequence was deposited in GenBank (accession number MH211598), and the closely related taxa were retrieved from the GenBank database using BLASTN software. The phylogenetic tree was constructed by MEGA 5.0 software according to the NeighborJoining (NJ) method. Voucher specimen (CCTCC no. M2018111) was deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China. Fermentation, Extraction and Isolation. The mycelium suspension of Streptomyces sp. N1510.2 was plated on Petri dishes containing 20 L Gause’s synthetic medium (20 mL/dish) (GAU; 20 g/L amylogen, 1 g/L KNO3, 0.5 g/L NaCl, 0.5 g/L K2HPO4·H2O, 0.5 g/L MgSO4·H2O, and 0.01 g/L FeSO4·H2O) containing potassium dichromate (100 μg/mL) and nalidixic acid (20 μg/mL). The plates were cultured for 7 days at 32 °C. Then, the culture medium was extracted with EtOAc four times. After the solvent was removed, the extract (7.8 g) was fractionated by silica gel CC and eluted using nhexane-acetone (10:1, 5:1, 2:1, 1:1, acetone) to give five fractions (F1−F5). Fraction F2 (300 mg, n-hexane-acetone 5:1) was further fractionated by silica gel CC eluting with n-hexane-acetone (8:1) to give subfractions F2a and F2b. Subfraction F2a (66 mg) was further purified by preparative HPLC eluting with CH3OH-H2O (70:30, v/v) to give 1 (12 mg). Subfraction F2b (42 mg) was further purified by preparative HPLC eluting with CH3OH-H2O (70:30, v/v) to give 3 (8 mg). Fraction F3 (410 mg, n-hexane-acetone 2:1) was isolated by silica gel CC eluting with n-hexane-acetone (6:1) to three subfractions F3a−F3c. Compound 2 (4.6 mg) was obtained from subfraction F3b by preparative HPLC eluting with CH3OH-H2O (70:30, v/v). Strepantibin A (1). Yellowish crystal, mp 150−152 °C; [α]20 D +7.4 (c 0.05, MeOH); IR (KBr) 3486, 3081, 3009, 1701, 1637, 1604, 1571,

(Figure 5). Compounds 1−3 also showed strong cytotoxicity to hepatoma carcinoma cells HepG-2, SMMC-7721, and plc-

Figure 5. Lineweaver−Burk plots for HK2 inhibition by strepantibitins A−C (1−3).

Table 2. Cytotoxicity of 1−3 against LO2, HepG-2, SMMC7721, and plc-prf-5 Cells (IC50, μM) cell lines 1 2 3 adriamycin gefitinib fluorouracil

LO2 11.2 17.6 17.8 13.5 19.8 10.6

± ± ± ± ± ±

HepG-2 0.5 0.7 0.6 0.4 0.4 0.3

2.3 12.3 9.5 2.6 6.6 3.6

± ± ± ± ± ±

0.3 0.4 1.3 0.4 0.2 0.2

SMMC-7721 2.9 4.9 10.3 1.1 9.2 2.5

± ± ± ± ± ±

0.8 1.1 2.0 0.3 0.3 0.2

plc-prf-5 1.9 11.6 7.1 1.9 4.0 2.8

± ± ± ± ± ±

0.2 0.6 0.5 0.1 0.2 0.2

prf-5 (Table 2). Strepantibin A (1) displayed the most potent HK2-inhibitory activity and cytotoxicity. Its cytotoxicity to HepG-2, SMMC-7721, and plc-prf-5 cells, with IC50 values 2.3, 2.9, and 1.9 μM, respectively, was similar to that of the positive control adriamycin (IC50 2.6, 1.1, and 1.9 μM). Additionally, strepantibin A had much lower cytotoxicity to normal human liver cells LO2, highlighting its potential as a lead compound. After treatment of SMMC-7721 cells with strepantibin A at concentrations ranging from 0.5 to 8.0 μM for 24 h, SMMC7721 cells resulted in chromatin dispersion and the formation of apoptotic bodies in DAPI staining test (Figure 6). The exact mechanisms associated with apoptosis induced by strepantibin A are under investigation.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer 341 polarimeter. UV spectra were

Figure 6. Fluorescence micrographs of strepantibin A-treated SMMC7721 (24 h) stained with DAPI. Magnification: 100×. D

DOI: 10.1021/acs.jnatprod.8b00821 J. Nat. Prod. XXXX, XXX, XXX−XXX

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1491 cm−1; UV (MeOH) λmax (log ε) 204 (2.91), 250 (2.45) nm; for 1 H and 13C NMR data, see Table 1; positive HR-ESI-MS m/z 349.1432 [M + H]+ (calcd for C22H21O4, 349.1440). Strepantibin B (2). Yellowish solid, mp 167−169 °C; IR (KBr) 3086, 3048, 1647, 1620, 1596, 1572, 1493 cm−1; UV (MeOH) λmax (log ε) 250 (4.43), 313 (4.85), 412 (3.48) nm; for 1H and 13C NMR data, see Table 1; positive HR-ESI-MS m/z 316.1325 [M + H]+ (calcd for C21H18NO2, 316.1338). Strepantibin C (3). Yellowish solid, mp 85−87 °C; IR (KBr) 3357, 3058, 3025, 1629, 1604, 1573, 1491 cm−1; UV (MeOH) λmax (log ε) 280 (2.81), 355 (2.36) nm; for 1H and 13C NMR data, see Table 1; positive HR-ESI-MS m/z 347.1310 [M + H]+ (calcd for C22H19O4, 347.1283); negative HR-ESI-MS m/z 345.1107 [M−H]+ (calcd for C22H17O4, 345.1127). X-ray Crystallographic Data of Strepantibin A (1). C22H20O4, M = 348.38 g/mol, Orthorhombic, space group P212121, a = 8.73540(10) Å, b = 10.3344(2) Å, c = 18.8954(3) Å, V = 1705.79(5) Å3, α = β = γ = 90°, Z = 4, T = 173(2), dcalcd = 1.357 mg/m3, μ = 0.752 mm−1, F(000) = 736. Graphite-monochromated Cu Kαave radiation λ = 1.54178 Å. 11 433 reflections measured (8.556° ≤ θ ≤ 140.294°), 2986 unique, which were used in all calculations. The final R1 was 0.0311 (I > 2 σ(I)) and wR2 was 0.0824 (all data). The absolute structure parameter was 0.04(5). Supplementary publication number CCDC 1864831. X-ray Crystallographic Data of Strepantibin B (2). C21H17NO2, M = 315.36 g/mol, Monoclinic, space group P21, a = 5.8252(6) Å, b = 13.4510(15) Å, c = 20.651(2) Å, V = 1617.7(3) Å3, α = 90°, β = 91.375(3)°, γ = 90°, Z = 4, T = 296(2) K, dcalcd = 1.295 mg/m3, μ = 0.083 mm−1, F(000) = 664. Graphite-monochromated Mo Kαave radiation λ = 0.71073 Å. 29 089 reflections measured (3.029° ≤ 2θ ≤ 27.101°), 2087 unique, which were used in all calculations. The final R1 was 0.0729 (I > 2 σ(I))and wR2 was 0.2061 (all data). Supplementary publication number CCDC 1865186. X-ray Crystallographic Data of Strepantibin C (3). C22H18O4, M = 346.36 g/mol, Monoclinic, space group C 2/c, a = 28.975(4) Å, b = 7.1541(8) Å, c = 21.497(3) Å, V = 3455.1(8) Å3, α = 90°, β = 129.162(7)°, γ = 90°, Z = 8, T = 296(2) K, dcalc = 1.332 g/cm3, μ = 0.091 mm−1, F(000) = 1456. Graphite-monochromated Mo Kαave radiation λ = 0.71073 Å. 64 385 reflections measured (2.82° ≤ 2θ ≤ 27.653°), 4015 unique, which were used in all calculations. The final R1 was 0.0703 (I > 2 σ(I)) and wR2 was 0.1350 (all data). Supplementary publication number CCDC 1865211. In Vitro Hexokinase II (HK2) Inhibition Assay.24 To screen for the strains with HK2 inhibiting activity and assay the inhibitory effects of isolated compounds on HK2 (Merck in China), the NADH generated through a coupled reaction with glucose-6-phosphate dehydrogenase (G6P-DH, Merck in China) was monitored at 340 nm. Ten microliters of HK2 (1 μM) was incubated with 5 μL of various concentrations of the culture extracts of actinomyces or strepantibins A−C (1−3) dissolved in deionized water at 37 °C for 10 min. Then, 85 μL of a mixture containing 100 mM Tris HCl pH 8.0, 5 mM MgCl2, 200 mM glucose, 0.8 mM ATP, 1 mM NAD+, and 0.25 units of G6P-DH was added. The enzyme inhibition IC50 values for each compound were determined based on the changes in absorbance at 340 nm. Triplicate experiments were conducted for each test. Lineweaver−Burk Plots for HK2 Inhibition. The Lineweaver− Burk plot experiments were performed to investigate the way of the compounds inhibiting HK2. Under the conditions of the most suitable for HK2 (37 °C, pH 7.3), different concentrations of substrates were added into 96 well plates and, based on the results of inhibition experiments, the compounds were added at 10 μM in each well. Through detection of the velocity of enzyme reaction, the formula of Lineweaver−Burk plots below was used to calculate the Km of HK2 with or without inhibitors, in which vm represented the maxvelocity of enzyme, S for concentration of substrate, and Km for Michaelis−Menten constant. All experiments were repeated three times independently.

Cell Viability Assay. Cell viability was measured using MTT assay as used in our previous research.25 Human normal liver cell LO2 and hepatoma carcinoma cells HepG-2, SMMC-7721, and plc-prf-5 were obtained from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (China). Briefly, the cells of LO2, HepG-2, SMMC-7721, and plc-prf-5 were seeded into 96-well culture plates (4−5 × 103 per well), respectively. After incubation for 24 h, the cells were treated with various concentrations of strepantibins A−C (1−3), and incubated for at 37 °C. Twenty microliters of MTT (5 mg/mL) was added to each well and incubated for another 4 h at 37 °C. After removal of the culture medium, the cells were lysed in 200 μL dimethyl sulfoxide (DMSO). Deionized H2O with the same DMSO concentration was used as negative control; adriamycin, gefitinib, and fluorouracil were used as the positive controls. Optical density was measured at 570 nm with a microplate reader (Model 550; Bio-Rad Laboratories, United States). The following formula was used to calculate cell viability: cell viability = (OD of the experimental sample/OD of the control group) × 100%. Triplicate experiments were conducted for each test. DAPI Staining.26 Cells were seeded on 12 mm round glass coverslips at a density of 4−5 × 104/mL in 24-well plates. After 24 h incubation, cells were treated with strepantibin A (1) (0.5−8.0 μmol/ L) for 48 h. After the cells were washed with cold PBS, cold methyl alcohol:acetone (1:1) was used to fix cells. Then, the cells were washed with PBS and stained with 4 μg/mL 4′,6-diamidino-2phenylindole (DAPI, Merck in China) for 10 min at room temperature. Coverslips containing the cells were then washed with PBS-TX (10 mL PBS + 10 μL 10% Triton X-100) three times and mounted using mounting medium (PBS:glycerol 1:1, v/v) and analyzed by fluorescence microscopy (Leica DM IRB).

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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.8b00821. Photographs of S. madraspatanum and its nest; the colony morphology, 16S rRNA gene sequences data and phylogenetic analysis of Streptomyces sp. N1510.2; the HRESIMS and NMR spectra of 1−3, the proposed biosynthesis pathway of 1 and 3 (PDF) Crystallographic information for Compound 1 (CIF) Crystallographic information for Compound 2 (CIF) Crystallographic information for Compound 3 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-631-5688303; Fax: 0086-631-5688303; E-mail: [email protected]. ORCID

Wei-Dong Xie: 0000-0003-2781-922X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of Shandong Province (Grant ZR2014HM018) and National Natural Science Foundation of China (Grant 31741048).

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REFERENCES

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K 1 1 = m + v vmS vm E

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