Nonocarbolines A–E, β-Carboline Antibiotics Produced by the Rare Actinobacterium Nonomuraea sp. from Indonesia

During the course of our ongoing screening for novel biologically active secondary metabolites, the rare Actinobacterium, Nonomuraea sp. 1808210CR was found to produce five unprecedented β-carboline derivatives, nonocarbolines A–E (1–5). Their structures were elucidated from high-resolution mass spectrometry, 1D and 2D nuclear magnetic resonance spectroscopy, and the absolute configuration of 4 was determined by using the modified Mosher method. Nonocarboline B (2) displayed moderate antifungal activity against Mucor hiemalis, while nonocarboline D (4) exhibited significant cytotoxic activity against the human lung carcinoma cell line A-549 with the IC50 value of 1.7 µM.


Introduction
Due to the serious consequence and dynamic nature of antibiotic resistance in pathogens, the need for new bioactive compounds is steadily increasing, especially regarding molecules with new modes of action [1]. In the past decades, actinobacteria, particularly from the genus Streptomyces, have been reported to produce about two thirds of the naturally derived antibiotics in current clinical use, as well as many anticancer compounds [2]. While Streptomyces species appear to have been exhaustively explored [3,4], other genera belonging to the so-called "rare actinobacteria" may still serve as promising sources for novel biologically active secondary metabolites [4][5][6][7]. One of these genera is Nonomuraea, which has recently been reported to produce several new biologically active compounds such as the antimicrobial hypogeamycins B-D [8], nonomuric acid and 3-hydroxy deoxydaunorubicinol aglycone [9], the cytotoxic hypogeamycin A [8], and karamomycins [10].
In the course of our screening for novel bioactive metabolites from our rare actinobacteria collections, the strain Nonomurea 1808210CR was selected for further investigation because it showed significant activity (MIC 4.2 µg/mL) against Bacillus subtilis and some potentially new metabolites were detected in the active samples by High Performance Liquid Chromatography coupled with diode 2 of 12 array detection and mass spectrometry (HPLC-DAD/MS). Herein, we describe the isolation, structure elucidation, and biological activities of nonocarbolines A-E (1)(2)(3)(4)(5), which constitute the first β-carbolines from this genus.

Results and Discussion
During our course for novel antibiotics from actinobacteria, significant activity by bioassay screening against Bacillus subtilis was detected in the extract of the strain 1808210CR. Phylogenetic 16S rRNA gene analysis showed that the aligned sequence was closely related to the DNA sequence of the type strain Nonomuraea jabiensis DSM 45507 T with 99.38% similarity (see Figures S1 and S2 in SI). The sequence was deposited in GenBank with the accession number MN 938364. The strain may represent a new species, and a polyphasic taxonomic study that will be reported elsewhere is presently ongoing.
Analysis of the crude extract by HPLC-DAD/MS followed by comparison with the Dictionary of Natural Products (DNP) database (http://dnp.chemnetbase.com) suggested the presence hitherto of unknown metabolites. Accordingly, we conducted a scale-up fermentation, and subsequent chromatography of the crude extract led to the isolation of five unknown β-carbolines ( Figure 1). Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 13 significant activity (MIC 4.2 µg/mL) against Bacillus subtilis and some potentially new metabolites were detected in the active samples by High Performance Liquid Chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD/MS). Herein, we describe the isolation, structure elucidation, and biological activities of nonocarbolines A-E (1)(2)(3)(4)(5), which constitute the first β-carbolines from this genus.

Results and Discussion
During our course for novel antibiotics from actinobacteria, significant activity by bioassay screening against Bacillus subtilis was detected in the extract of the strain 1808210CR. Phylogenetic 16S rRNA gene analysis showed that the aligned sequence was closely related to the DNA sequence of the type strain Nonomuraea jabiensis DSM 45507 T with 99.38% similarity (see Figure S1-S2 in SI). The sequence was deposited in GenBank with the accession number MN 938364. The strain may represent a new species, and a polyphasic taxonomic study that will be reported elsewhere is presently ongoing.
Analysis of the crude extract by HPLC-DAD/MS followed by comparison with the Dictionary of Natural Products (DNP) database (http://dnp.chemnetbase.com) suggested the presence hitherto of unknown metabolites. Accordingly, we conducted a scale-up fermentation, and subsequent chromatography of the crude extract led to the isolation of five unknown β-carbolines ( Figure 1).   [11,12]. The complete structure of 1 was determined by 1D and 2D NMR analyses. The 13 Figure S18 in SI). The 1 H, 1 H COSY (correlation spectroscopy) correlations ( Figure S21 in SI), in conjunction with the 1 H, 13 C HMBC correlations ( Figure S23 in SI), assembled the 1,3-disubstituted β-carboline moiety. Furthermore, the COSY spectrum showed a series of correlations from H-12 to H-13, H-13 to H-14, H-14 to H-15, and H-15 to H-16 (see Table 1), indicating the presence of a contiguous pentyl chain, which was connected to C-1 via carbonyl C-11 from an HMBC (Heteronuclear Multiple Bond Correlation) correlation of H-13 to C-11. Since there is no direct observation of 3 JCH coupling in HMBC correlations from H-12 to C-1, we   [11,12]. The complete structure of 1 was determined by 1D and 2D NMR analyses. The 13 C NMR data confirmed the presence of 18 carbons, including one carboxylic acid (δ C 166.3), one ketone (δ C 203.1), five olefinic methines (δ H 7.35-9.14; δ C 113.3-129.2), six nonprotonated carbons, including four carbons attached to a heteroatom (δ C 120.2-142.2), four methylene carbons (δ H 1.37-3.41; δ C 22.0-36.7), and one methyl group (δ H 0.90; δ C 13.6) (see Figures S19, S20 and S22 in SI). In addition, the 1 H NMR spectrum of 1 in DMSO-d 6 provided an NH signal at 12.27 ppm ( Figure S18 in SI). The 1 H, 1 H COSY (correlation spectroscopy) correlations ( Figure S21 in SI), in conjunction with the 1 H, 13 C HMBC correlations ( Figure  S23 in SI), assembled the 1,3-disubstituted β-carboline moiety. Furthermore, the COSY spectrum showed a series of correlations from H-12 to H-13, H-13 to H-14, H-14 to H-15, and H-15 to H-16 (see Table 1), indicating the presence of a contiguous pentyl chain, which was connected to C-1 via carbonyl C-11 from an HMBC (Heteronuclear Multiple Bond Correlation) correlation of H-13 to C-11. Since there is no direct observation of 3 J CH coupling in HMBC correlations from H-12 to C-1, we compared the carbon chemical shifts of the isolated compounds with those of marinacarboline B (8), Antibiotics 2020, 9, 126 3 of 12 which has a similar basic skeleton. The carbon chemical shift at C-1 of the isolated compound 1 was identical with the published data [13]. Moreover, an NOE (Nuclear Overhauser Effect) correlation between the NH and H-12 supported the location of the carbonyl side chain at C-1. Further HMBC correlations from H-4 to the carbonyl carbon C-10, together with the molecular formula, indicated that the carboxylic acid was attached to C-3. Therefore, the structure of compound 1, for which we propose the trivial name nonocarboline A, was unambiguously determined as 1-hexanoyl-9H-pyrido [3,4-b] indole-3-carboxylic acid.
Nonocarboline B (2) was isolated as a yellow solid and its molecular formula was determined as C 19 H 20  The molecular formula of nonocarboline D (4), which was isolated as a yellow solid, was determined by its HR-ESIMS to be C 19 H 20 N 2 O 4 (11 DBE) (Figures S12-S14 in SI). The UV, mass and NMR spectra were very similar to those of 3, indicating that 4 represents a structural isomer of 3. The COSY spectrum showed a contiguous aliphatic chain from H-12 to H-17, bearing a hydroxyl group at C-16 due to the deshielded shift of proton signal at 3.59 ppm with the corresponding carbon at 65.7 ppm (see Figures S35-S40 in SI). The absolute configuration of 4 was determined by the modified Mosher method by esterification using (R)-and (S)-MTPA chloride to provide (S)-and (R)-MTPA esters [14,15]. The shift differences ∆δ S-R calculated between these esters (see Figures S47-S51 in SI) are depicted in Figure 2. The absolute configuration of 4 was determined to be R.
Antibiotics 2020, 9, x FOR PEER REVIEW 3 of 13 compared the carbon chemical shifts of the isolated compounds with those of marinacarboline B (8), which has a similar basic skeleton. The carbon chemical shift at C-1 of the isolated compound 1 was identical with the published data [13]. Moreover, an NOE (Nuclear Overhauser Effect) correlation between the NH and H-12 supported the location of the carbonyl side chain at C-1. Further HMBC correlations from H-4 to the carbonyl carbon C-10, together with the molecular formula, indicated that the carboxylic acid was attached to C-3. Therefore, the structure of compound 1, for which we propose the trivial name nonocarboline A, was unambiguously determined as 1-hexanoyl-9Hpyrido [3 ,4-b]indole-3-carboxylic acid. Nonocarboline B (2) was isolated as a yellow solid and its molecular formula was determined as C19H20N2O3 by the molecular ion cluster [M+H] + at m/z 325.1548 (calcd. 325.1552) in its HR-ESIMS spectrum (see Figures S5-S8 in SI). Compared to 1, the molecular formula of 2 includes an additional CH2 moiety. The 1 H and 13 C NMR spectral data (Figures S24-S29 in SI) of 2 were similar to those of 1, except that the signal at δH 0.90 ppm (3H, t, 7.0 Hz) of 1 was replaced by an isopropyl signal (6H, d, 6.5 Hz), indicating the presence of an isohexyl chain connected to C-1 through the carbonyl C-11. Nonocarboline C (3) was obtained as a yellow solid and exhibited a molecular ion peak at m/z 341.1495 (calcd. 341.1501) which indicated the molecular formula of C19H20N2O4 and 11 degrees of unsaturation (Figures SS9-S11 in SI). The main difference in the 1 H NMR spectrum of 3 compared to 2 is the disappearance of the proton signal of H-15. The presence of a hydroxyl group at C-15 was confirmed in the 13 C NMR spectrum by the presence of a deshielded shifted signal of 68.8 ppm at C- The molecular formula of nonocarboline D (4), which was isolated as a yellow solid, was determined by its HR-ESIMS to be C19H20N2O4 (11 DBE) (Figures S12-S14 in SI). The UV, mass and NMR spectra were very similar to those of 3, indicating that 4 represents a structural isomer of 3. The COSY spectrum showed a contiguous aliphatic chain from H-12 to H-17, bearing a hydroxyl group at C-16 due to the deshielded shift of proton signal at 3.59 ppm with the corresponding carbon at 65.7 ppm (see Figures S35-S40 in SI). The absolute configuration of 4 was determined by the modified Mosher method by esterification using (R)-and (S)-MTPA chloride to provide (S)-and (R)-MTPA esters [14,15]. The shift differences Δδ S-R calculated between these esters (see Figures S47-S51 in SI) are depicted in Figure 2. The absolute configuration of 4 was determined to be R. HR-ESIMS analysis of nonocarboline E (5) revealed a molecular ion peak at m/z 383.1608 (calcd. 383.1607) with the molecular formula C21H22N2O5 (12 DBE) (Figures S15-S17 in SI). The 1 H NMR spectrum of 5 contained all signals of nonocarboline B (2) with additional resonances for a methylene at 3.84 and 3.92 (δC 68.5) and an sp 2 carbon (δC 170.5). Compared to 2, the structure of 5 included an acetoxy group which was connected to the methylene H-16 from HMBC correlations of H-16 to C-17, and of methyl-18 to C-17 (see Figures S41-S46 in SI).
The first isolated β-carboline alkaloid was harmaline in 1841 from the plant Peganum harmala [16] and since then, numerous compounds have been isolated from diverse sources, such as dichotomines A-D from plants [11], gibellamines A and B from fungi [17], and marinacarbolines A-D from bacteria  The first isolated β-carboline alkaloid was harmaline in 1841 from the plant Peganum harmala [16] and since then, numerous compounds have been isolated from diverse sources, such as dichotomines A-D from plants [11], gibellamines A and B from fungi [17], and marinacarbolines A-D from bacteria [13]. Interestingly, the synthesis of a methyl ester form of 1 was reported by Chalotra et al. [18]. β-carbolines are known to exhibit a variety of biological activities, including antimicrobial [19,20], antitumor [21,22], antiparasitic [23,24], and antiviral effects [25]. According to our literature survey, 1,3-disubstituted β-carboline derivatives bearing a carbonyl moiety at C-1 and a carboxyl group at C-3 are known from plants, sponges, and fungi, while their amide-bearing analogues were reported from plants and bacteria. Of these, we show in Figure 3 stellarine A (6) from the marine-derived fungus Dichotomomyces cejpii F31-1 [26], cestrumine B (7) from the plant Cestrume hediundinum [27], and marinacarboline B (8) from the marine actinobacterium Marinactinospora thermotolerans [13].
The analogue JBIR 133 (9) from Kitasatospora setae [28] also contains a carboxylic acid group at C-3, but not at C-1 ( Figure 3). However, the current study is the first report on the occurrence of βcarboline derivatives possessing a carboxylic acid at the C-3 position and a carbonyl moiety at C-1 that have been isolated from bacteria.
Studies on the biosynthesis of bacterial β-carbolines have so far been limited because only a few compounds have been isolated from these organisms. A recent study showed that the biosynthetic gene McbB from Marinactinospora thermotolerans was responsible for the biosynthesis of marinacarbolines via a Pictet-Spengler condensation of L-tryptophan and oxaloacetate [29]. Posssibly, nonocarbolines A-E are also biosynthesized in the same way by condensation of Ltryptophan and oxaloacetate, followed by chain elongation, hydroxylation, or esterification to form the final structure of nonocarbolines A-E. However, feeding studies with labeled precursors and/or elucidation of the corresponding gene cluster in our strain remain necessary to prove this hypothesis.   The analogue JBIR 133 (9) from Kitasatospora setae [28] also contains a carboxylic acid group at C-3, but not at C-1 ( Figure 3). However, the current study is the first report on the occurrence of β-carboline derivatives possessing a carboxylic acid at the C-3 position and a carbonyl moiety at C-1 that have been isolated from bacteria.
Studies on the biosynthesis of bacterial β-carbolines have so far been limited because only a few compounds have been isolated from these organisms. A recent study showed that the biosynthetic gene McbB from Marinactinospora thermotolerans was responsible for the biosynthesis of marinacarbolines via a Pictet-Spengler condensation of L-tryptophan and oxaloacetate [29]. Posssibly, nonocarbolines A-E are also biosynthesized in the same way by condensation of L-tryptophan and oxaloacetate, followed by chain elongation, hydroxylation, or esterification to form the final structure of nonocarbolines A-E. However, feeding studies with labeled precursors and/or elucidation of the corresponding gene cluster in our strain remain necessary to prove this hypothesis.
Nonocarbolines A-E were evaluated for antimicrobial and cytotoxic activities. Compounds 1-4 showed weak to moderate activity against Bacillus subtilis, whereas compound 5 was not active. Compound 2 was the most active derivative in our antimicrobial test panel against Mucor hiemalis with a minimum inhibitory concentration (MIC) at 8.3 µg/mL. Moreover, a cytotoxicity assay was conducted against six different cancer cell lines (see Table 2), and compound 4 was found to be the most active one against human lung carcinoma A-549 with an IC 50 value of 1.7 µM, while the other metabolites showed very weak or no cytotoxic effects.

Sampling and Isolation of the Organism
A soil sample was collected from Malang, East Java, Indonesia. One gram of sample was heated at 60 • C for 30 min to eradicate all the vegetative cells that were present in the sample. Ten milliliters of sterile water were added to the samples, and the mixture was serially diluted (1:10, 1:100, and 1:1000). The sample was transferred on agar medium 5336 (soluble starch (10 g/L), casein (peptone Typ M) (1 g/L), K 2 HPO 4 (0.5 g/L), MgSO 4 × 7H 2 O (5.0 g/L), and agar (20 g/L)). The pH was adjusted to 7.3 before sterilization and supplemented with cycloheximide (100 µg/mL) as an antifungal agent [30] and incubated for 7-21 days at 30 • C.
The PCR product was checked on the agarose gel (0.8%) and purified using the NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol. DNA sequencing was performed by using a 96-capillary system from Applied Biosystems (ABI), 3730xl DNA Analyzer. The primers for sequencing were F27, R518, F1100, R1100, and R1492. The 16S rRNA gene sequence was edited, and the contig was assembled and generated by BioEdit software (version 7.0.5.3) (company city country) [32]. The 16S rRNA gene sequence was deposited in GenBank with the accession number MN938364.
Identification of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities were carried out using EzTaxon-e server (http://www.ezbiocloud.net/taxonomy) [33] and the sequences of the strains were aligned using the CLUSTAL W algorithm [33] from the MEGA X software package version 10.0.5 for Windows (MEGA X, Penn State University, Pennsylvania, USA) [34]. Phylogenetic analysis was conducted using neighbor-joining [35] algorithms from MEGA X. The evolutionary distances were computed using the Kimura 2-parameter method [36]. The topologies of the inferred trees were evaluated by bootstrap analyses [37] based on 1000 replicates.

Scale-up Fermentation, Extraction and Isolation
A well-grown culture on an agar plate (containing glucose 4 g, yeast extract 4 g, malt extract 10 g, CaCO 3 10 g, agar 12 g in 1 L of deinonized water, pH adjusted to 7.2 before sterilization) was cut into small pieces (1 cm) and three pieces per flask were inoculated in a batch of thirty 250 mL Erlenmeyer flasks containing 100 mL of the medium composed of 15 g of glucose, 15 g of soybean meal, 5 g of corn steep liquor, 2 g of CaCO 3 and 5 g of NaCl in 1 L distilled water, pH was adjusted to 7.0 before sterilization. The cultures were incubated at 37 • C on a rotary shaker (120 rpm). The strain growth was monitored by constant checking of the amount of free glucose (using Medi-Test, Macherey Nagel). The fermentation was stopped 5 days after glucose depletion. In total, 18 L of fermentation were produced in 6 batches (3 L each batch). The mycelial cake was separated from the supernatant by centrifugation (3000 rpm, 10 min). The biomass was extracted with ethyl acetate three times (1.5 L) in an ultrasonic bath at 40 • C for 30 min. After filtration and evaporation, the residue was redissolved in MeOH/H 2 O (7:3) and partitioned with n-heptane to remove the lipophilic components. The methanol layer was

Antimicrobial Assay
Minimum inhibitory concentrations were determined by a serial dilution assay in 96-well plates according to our standard protocols [38]. Twenty microliter aliquots of compounds 1-5 with an initial concentration of 1 mg/mL (the final concentration in the first well is 67 µg/mL) were tested against three different Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus Newman, and Mycobacterium smegmatis), five Gram-negative bacteria (Acinetobacter baumanii, Citrobacter freundii, Escherichia coli wild type, Escherichia coli strain acrB, and Pseudomonas aeruginosa) and three fungi (Candida albicans, Mucor hiemalis and Pichia anomala,) with methanol as a negative control. Oxytetracycline, ciprofloxacin, and kanamycin were used as positive controls against Gram-positive and Gram-negative bacteria, whereas nystatin was used against fungi.

Cytotoxicity Activity
Cytotoxicity (IC 50 ) of compounds 1-5 was determined against seven human cancer cell lines by using an MTT assay according to an established procedure [39]. The cell lines were cultured in DMEM (Gibco, ThermoFisher Scientific, Hilden, Germany) and RPMI media (Lonza, Cologne, Germany) for MCF-7. All cell lines were supplemented with 10% fetal bovine serum (Gibco) and incubated under 10% CO 2 at 37 • C. Epothilone B was used as a positive control and methanol as a negative control.

Conclusions
The current study shows that rare Actinobacteria are still promising sources for novel bioactive metabolites, since five new β-carboline alkaloids have been discovered from a strain that probably represents a new species of the rare and underexploited genus Nonomuraea. Even though the preliminary biological characterization of the novel molecules did not give any hints on their potential for drug development because their effects in biological systems were rather moderate and not particularly selective, the outcome of this work should give encouragement to continue the search for novel producer strains, in particular in hitherto underexploited geographic areas like Indonesia.
Author Contributions: G.P. is highly indebted to DAAD-GINAICO program for the PhD scholarship and The President's Initiative and Networking Funds of the Helmholtz Association of German Research Centres (German: Helmholtz Gemeinschaft Deutscher Forschungszentren or HGF) under Contract Number VH-GS-202. We thank Christel Kakoschke for recording the NMR experiments, Wera Collisi for conducting cytotoxicity assays, Aileen Gollasch and Klaus-Peter Conrad and Silke Reinecke for technical assistance, and Hedda Schrey for fruitful discussion.
Funding: J.W. and M.S. were supported by a grant (GINAICO, 16GW0105) of the German Ministry for Education and Research (BMBF). G.P. was supported by a PhD scholarship funding program from DAAD-GINAICO number 57342738 (91621443).