Special Collection of Papers: Reviews
Bioactive Secondary Metabolites with Unique Aromatic and Heterocyclic Structures Obtained from Terrestrial Actinomycetes Species
2016 Volume 64 Issue 7 Pages 668-675
Details
2016 Volume 64 Issue 7 Pages 668-675
Natural products from actinomycetes are important and valuable sources for drug discovery and the development of biological tools. The present review describes our recent study on the isolation of new natural products mainly possessing heterocyclic and aromatic ring structures with biological effects on cancer-related cellular pathways such as tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) and Wnt signaling.
Actinomycetes from different sources are widely recognized to produce secondary metabolites with various biological activities.1,2) During our studies on the search for bioactive secondary metabolites from various natural resources such as plants3) and myxomycetes4) and other microorganisms, we have developed a particular interest in a screening program for new natural products from actinomycetes5) isolated from soil samples collected in various locations in Japan, mainly from Chiba Prefecture, using in-house biological assays targeting cancer-related pathways such as tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL)6) and Wnt7) signaling.
TRAIL is a member of the tumor necrosis factor (TNF) family of apoptosis-triggering proteins.8) TRAIL-induced apoptosis is initiated when it binds to its receptors (TRAIL-Rs).9) Four homologous human TRAIL-Rs have been reported to date, and are called TRAIL-R1 (also called DR4), TRAIL-R2 (DR5, Apo2, TRICK2, or KILLER), TRAIL-R3 (DcR1/TRID), and TRAIL-R4 (DcR2). The death receptors (DRs), DR4 and DR5 comprise a conserved death domain (DD) motif and are responsible for the induction of apoptosis. Treatments with TRAIL have become a new strategy for cancer because it selectively induces apoptosis in cancer cells, while having negligible to no effects on normal cells.10) However, some malignant tumors such as gastric, breast, lung, prostate, colon, and other cancer cells are resistant to apoptosis by TRAIL. Dysfunction of death receptors DR4 and DR5, over expression of cell survival proteins such Bcl-2 and Bcl-X, and loss of apoptosis inducers such as Bak and Bax, have been reported to result in TRAIL-resistance. Therefore, the search for agents with the ability to overcome TRAIL resistance may contribute to the development of effective anticancer drugs.
On the other hand, Wnt signaling plays a key role in many biological events including cell morphology, motility, proliferation, and differentiation.11) However, aberrant Wnt/β-catenin signaling may lead to the formation of tumors. Therefore, small molecules that regulate Wnt signaling may help to cure various diseases. In our laboratory, a screening program using a cell-based luciferase assay was performed in order to evaluate T-cell factor (TCF)/β-catenin transcriptional activity (TOP assay).12)
The present review attempts to summarize the chemical structures of compounds mainly isolated by our group from actinomycetes along with their biological activities. We divided the isolated compounds into six categories based on their chemical structures.
The strain Streptomyces sp. IFM 11490 was obtained from a soil sample collected from the main gate area of Hokkaido University, Sapporo, Japan. The crude extract of the culture of this strain exhibited cytotoxicity against human gastric adenocarcinoma (AGS) cells and also showed a number of colorful spots in a TLC examination with anisaldehyde and sulfuric acid spray reagents. The liquid culture of this strain in Waksman medium at 28°C for 6 d with shaking at 200 rpm was harvested and extracted with EtOAc to give an extract that was then subjected to fractionation by chromatography to yield three new isobenzofuran derivatives: elmonin (1) and elmenols A (2) and B (3),13) as well as the known angucyclinone X14881E (4).14) Elmonin (1) with the molecular formula C20H16O4 represents an interesting carbon skeleton containing a spiro[isobenzofuran-1,2′-naptho[1,8-b,c]furan] moiety, which was elucidated on the basis of detailed spectroscopic data. The spectral data of elmonin (1) proved to be identical to those of oleaceran,15) which was isolated from Streptomyces sp. Lv20195 cultivated from the root zone of Olea europea by Müller and colleagues based on its interesting chemical profiling. The structure of oleaceran was determined by detailed spectral analysis. The optical rotation value of elmonin (1) in methanol was levorotatory, while that of oleaceran in methanol was dextrorotatory, suggesting that they are enantiomers. We used the name “elmonin” in our patent,16) which was applied for (application date: March 28, 2013) earlier than the study on oleaceran15) was submitted (on May 27, 2013) and published on the Web (on June 26, 2013). Elmonin and oleaceran have one chiral center, while their absolute configurations have not yet been determined.
Elmenols A (2) and B (3), having the same molecular formula of C21H20O4, were found to be epimers with an 1,3-dihydroisobenzofuran derivatives linked to an α-naphthol moiety. The relative configuration of 2 was determined in a single crystal X-ray diffraction study using CuKα radiation. The crystallographic data of 2 was collected for a single crystal (0.080×0.080×0.080 mm) at a temperature of −180.0±1°C. The crystal was orthorhombic, space group P212121 (#19) with a=7.8649(2) Å, b=11.0788(3) Å, c=19.7695(5) Å, and Z=4. An X-ray structural analysis of 2 established the relative positions of H-9 and H-16 as trans, as shown in Fig. 1. Although compounds 1–3 did not show TRAIL resistance-overcoming activity in AGS cells, elmonin (1) and elmenol B (3) were moderately cytotoxic against AGS cells (IC50, ca. 50 µM).
Elmonin (1) and elmenols A and B (2 and 3) may be biogenetically classified as members of a group of angucyclines, the polycyclic aromatic polyketides.17) Elmonin (1) possesses basically the same carbon framework as that of emycin E (5), which was obtained from a mutant strain of Streptomyces cellulosae ssp. griseoincarnatus 1114-2 in 1995 by Rohr and colleagues.18) Several isobenzofuran analogues related to angucyclines were recently isolated from Actinomycetes strains by other groups. Xie et al. reported the isolation of a 1,3-dihydroisobenzofuran derivative from marine Streptomyces sp. W007 in 2012,19) and also isolated an epimer of the 1,3-dihydroisobenzofuran in 2015 (=2015-isomer).20) The first 1,3-dihydroisobenzofuran isolated in 2012 (=2012-isomer) was initially proposed to have a cis H-9/H-16 configuration.19) However, they reassigned the H-9/H-16 configuration of the 2012-isomer as trans in 2015 (6) and that of the 2015-isomer as cis (7), respectively,20) and their absolute configurations were also proposed on the basis of electronic circular dichroism (CD) spectra along with time-dependent density functional theory (TDDFT) calculations.20) In 2015, Shen and colleagues also isolated a compound with identical spectral data to Xie’s 2012-isomer (6) from Streptomyces sp. CB01913 and suggested that the H-9/H-16 configuration of the 2012-isomer had to be revised as trans.21) The strain Streptomyces sp. CB01913 was isolated from a soil sample collected in Weishan County, Yunnan Province, China, and Shen and colleagues also isolated 16 compounds belonging to the angucycline and angucyclinone classes of natural products including six isobenzofuran metabolites (6–11) (Fig. 2). Elmenols A (2) and B (3) correspond to the dehydration products of compounds 6 and 7, respectively.
A hypothetical biosynthetic pathway (Fig. 3) for elmonin (1) and elmenols A (2) and B (3) may begin with the Baeyer–Villiger oxidation of X14881E (4) on C8/C9 and C16/C17 to give the lactone intermediates 4A and D, respectively. The precursor X14881E (4) was isolated from the extract of Streptomyces sp. IFM 11490. Rohr and colleagues18) reported that ochromycinone acted as a substrate for Baeyer–Villiger oxidation in the biosynthesis of emycin F (5). The intermediates 4A and D were opened by water hydrolysis to give 4B and E. The reduction and intramolecular cyclization of the intermediates produced led to the formation of elmonin (1) and elmenols A (2) and B (3).
The phenazine derivatives izumiphenazines A–D (12–15)22,23) along with the known phenazine-1,6-dicarboxylic acid,24) 1-hydroxyphenazine,25) phenazine-1-carboxylic acid,26) and 6-hydroxyphenazine-1-carboxylic acid27) were isolated from the ethyl acetate extract of Streptomyces sp. IFM 11204 (Fig. 4). Three glycoconjugated phenazines designated as izuminosides A–C (16–18) were also obtained from the culture extract of Streptomyces sp. IFM 11260.28) These two strains (Streptomyces spp. IFM 11204 and IFM 11260) were isolated from soil samples collected in Izumi Forest, Chiba, Japan. Compounds 13 (30 µM), 14 (20 µM), 17 (10 µM), and 18 (60 µM) in combination with TRAIL exhibited synergistic activities in the sensitization of TRAIL-resistant AGS cells. Yorophenazine (19), a phenazine-1,6-dicarboxylic acid linked to N-acetylcysteine, was isolated from Streptomyces sp. IFM 11307.29) This strain was isolated from a soil sample collected from Yoro-keikoku, Ichihara, Chiba, Japan. Compound 19 did not show any detectable reduction in cell viability with TRAIL.
The cultured Streptomyces sp. IFM 11694, collected from Namegata, Ibaraki, Japan, was found to produce one hydrophenazine derivative named aotaphenazine (20) and 5,10-dihydrophencomycin (21).30) The known phenazine-1-carboxylic acid (22),26) phencomycin (23),31) and phenazine-1,6-dicarboxylic acid (24)24) were also isolated (Fig. 5). Aotaphenazine (20) had a carboxylic anhydride moiety linked to a phenyl ring. Its structure was confirmed using gradient-enhanced heteronuclear multiple bond correlation (g-HMBC) and constant time inverse-detected gradient accordion rescaled heteronuclear multiple bond correlation (CIGAR-HMBC) techniques.32) The most important correlations observed from the CIGAR-HMBC experiment were the couplings of H-5 to C-3 (δC 165.3) and H-6 to C-1 (δC 161.5) through 5J coupling.
Arrows indicate key correlations by the CIGAR-HMBC experiment.
Aotaphenazine (20), similar to other phenazine derivatives, is formed biosynthetically via the shikimic acid pathway, as shown in Fig. 6. Previous studies showed that shikimic acid is converted to chorismic acid in known transformation steps that are part of the aromatic amino acid biosynthetic pathway.33,34) Chorismic acid is converted to the phenazine precursors by a series of enzymatically controlled steps to produce trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA).35) Two molecules of DHHA are converted into 5,10-dihydrophenazine-1,6-dicarboxylic acid. The latter may undergo mono-O-methylation to produce 21 and decarboxylation to give 5,10-dihydrophenazine-1-dicarboxylic acid. This then couples to the C6–C2 building block (i.e., phenylacetic acid) to produce aotaphenazine (20). In the same manner Abdel-Mageed et al.36) proposed a biosynthetic pathway for dermacozines E–G (25–27) possessing very similar structures to aotaphenazine (20).
Aotaphenazine (20) exhibited TRAIL resistance-overcoming activity in AGS cells at a concentration of 12.5 µM and up-regulated DR4 and DR5 protein levels. Furthermore, aotaphenazine (20) down-regulated the levels of the cell survival protein Bcl-2 in a dose-dependent manner. Sulfotanone (28) and panosialin wA (29)37) were also isolated from the mycelium of the same strain (Fig. 7). Compound 28 was assigned as 2-hydroxy-21-methyl-3-oxo-docosane-1-sulfonic acid by using a combination of spectroscopic techniques, including high resolution-electrospray ionization (HR-ESI)-MS, IR, and one and two dimensional (1 and 2D)-NMR measurements. Very few secondary metabolites containing sulfonic acids or sulfate esters from actinomycetes have been reported to date.38,39) Compound 28 is one of the unique alkyl sulfonic acid derivatives isolated from actinomycetes. To the best of our knowledge, sulfotanone (28) is one of the rare sulfonic acid containing natural products produced by actinomycetes. The absolute configuration of 28 was determined to be the S configuration using a modified Mosher’s method. Our results with TRAIL resistance-overcoming activity in AGS cells showed that compounds 28 and 29 exhibited activities at concentrations of 40.0 and 80.0 µM, respectively.
The acridine alkaloids inubosins A–C (30–32)40) were isolated from the culture broth of Streptomyces sp. IFM 11440 by bioassay-guided fractionation using a cell-based assay of neurogenin2 (Ngn2) promoter activity. This strain was obtained from a soil sample collected at Inubosaki Cape, Choshi, Chiba Prefecture, Japan. Ngn2 is an activator-type basic helix-loop-helix (bHLH) protein that may promote neural stem cell differentiation. Inubosin B (31) was found to exhibit the strongest Ngn2 promoter activity, showing 1.9-fold more activity at 20 µM than that of the positive control (baicalin, 100 µM), and enhanced the mRNA expression of relevant genes to neural stem cell differentiation.
Inohanamine (33) was produced by Streptomyces sp. IFM 11584. The strain was isolated from a soil sample collected at Inohana Park in Chiba, Japan. This compound was isolated by the “target protein oriented natural products isolation” (TPO-NAPI) method using the Hes1-immobilized beads constructed in our group.41) Hes1 belongs to repressor-type bHLH proteins and inhibits the expression of activator-type bHLH factors. Therefore, Hes1 inhibitors are expected to accelerate neural stem cell differentiation. Inohanamine (33) had the ability to bind to the Hes1 protein and possessed a 4-hydroxypiperidine ring linked to a side chain with three conjugated double bonds (Fig. 8).
The fermentation of Streptomyces sp. IFM 11307 isolated from a soil sample collected from Yoro Valley, Ichihara, Chiba, Japan gave four pyranonaphthoquinones (34–36).29) Compound 35 at 0.1 µM significantly overcame TRAIL resistance in AGS cell lines. The same Streptomyces species also yielded yoropyrazone (38).42) Its proposed structure contained a naphthoquinone moiety fused with a pyridazone ring. Yoropyrazone (38) exerted cytotoxic effects against AGS cells with IC50=12.5 µM. When compound 38 was combined with TRAIL, a synergistic effect was observed at 10.0 µM on AGS cells.
Katorazone (39),43) an alkaloid with a 2-azaanthraquinone-phenylhydrazone residue, was isolated from a culture of Streptomyces sp. IFM 11299 along with the known 1-hydroxy-6-methoxy-8-methyl-anthraquinone,44) utahmycin A (40),45) aloesaponarin II,44) and anthranilic acid. Utahmycin A (40) was obtained for the first time from a wild-type strain. Compound 39 in combination with TRAIL at 40.0 µM displayed a synergistic effect on AGS cells (Fig. 9).
Fuzanins A–I (41–49)46,47) are nine natural carbamate or pyridine derivatives that have been isolated from a culture of Kitasatospora sp. IFM 10917. This strain was isolated from a soil sample collected at Toyama Castle Park in Toyama, Japan. The absolute configurations of fuzanins A (41), B (42), and E–H (45–48) were determined using a modified Mosher’s method. Fuzanin D (44) moderately inhibited Wnt signal transcription at 25.0 µM and was also cytotoxic against DLD-1 cells (IC50=41.2 µM). A culture of Streptomyces sp. IFM 10937 yielded two tyrosine derivatives (50, 51)48) and the known novobiocin. The absolute configurations of 50 and 51 were determined by CD spectra and confirmed by Marfey’s method. Compound 50 (150.0 µM) in combination with TRAIL exhibited synergistic activity in the sensitization of TRAIL-resistant AGS cells (Fig. 10).
Screening for inhibitors of Wnt signaling in our group led to the isolation of different secondary metabolites from actinomycetes. The known nonactin (52), monactin (53), and dinactin (54) were isolated from the actinomycete CKK179.49) The dimeric dinactin (55) was also obtained from the culture of Streptomyces sp. (YM09-028). Compounds 52–55 were found to inhibit TCF/β-catenin transcriptional activity with IC50 values of 0.6–7.4 nM (Fig. 11).
The fermentation of actinomycete CKK748 gave griseoviridin (56), cyclo(4R-hydroxy-L-Leu-D-Pro) (57), and cyclo(4R-hydroxy-L-Phe-D-Pro) (58), while nocardamine (59), dehydroxynocardamine (60), desmethylenylnocardamine (61), bisucaberine (62), and the N-formylantimycic acid methyl ester (63) were isolated from a culture of actinomycete CKK784.50) Compounds 56, 59, and 60 inhibited TCF/β-catenin transcriptional activity with IC50 values of 8.2–14 µM (Fig. 12).
We herein reviewed our search for new bioactive natural products from actinomycetes with an emphasis on their chemical structures and biological activities. Most isolated compounds possess heterocyclic and aromatic ring systems such as isobenzofuran or angucycline (a polycyclic aromatic polyketide), phenazine, acridine, piperidine pyranonaphthoquinone or anthraquinone, cyclic carbamate, and pyridine. Many of the isolated compounds described here exert their effects on the TRAIL and Wnt pathways, and are expected to become new strategies in disease and development studies.
We thank Professor Tohru Gonoi (Medical Mycology Research Center, Chiba University) for the identification and deposit of actinomycete strains. This work was supported by KAKENHI Grant No. 23102008 on Innovative Areas, “Chemical Biology of Natural Products” from the Ministry of Education, Culture, Sports, Science and Technology of Japan, KAKENHI Grant Nos. 26305001 and 25870128 from the Japan Society for the Promotion of Science, and by the Uehara Memorial Foundation, Japan. Dr. M. S. Abdelfattah thanks JSPS for the Long-Term Invitation Fellowship for Research in Japan (L14567).
The authors declare no conflict of interest.
