Deuterium-Labeled Precursor Feeding Reveals a New pABA

Article pubs.acs.org/jnp

Deuterium-Labeled Precursor Feeding Reveals a New pABAContaining Meroterpenoid from the Mango Pathogen Xanthomonas citri pv. mangiferaeindicae Hesham Saleh,† Daniel Petras,† Andi Mainz,† Dennis Kerwat,† Ayse Nalbantsoy,‡ Yalcin Erzurumlu,§ and Roderich D. Süssmuth*,† †

Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova Izmir, Turkey § Department of Biochemistry, Faculty of Pharmacy, Ege University, 35100 Bornova Izmir, Turkey ‡

S Supporting Information *

ABSTRACT: A new para-aminobenzoic-acid-containing natural product from the mango pathogenic organism Xanthomonas citri pv. mangiferaeindicae is described. By means of stable isotope precursor feeding combined with nontargeted LC-MS/MS, the generated spectra were clustered and visualized in a molecular network. This led to the identification of a new member of the meroterpenoids, termed xanthomonic acid, which is composed of an isoprenylated para-aminobenzoic acid. In vitro cytotoxicity assays demonstrated activity of xanthomonic acid against several human cancer cell lines by induction of autophagy.

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he genus Xanthomonas comprises a number of plant pathogens with significant impact on various crops, such as sugar cane, soybean, wheat, and mango. In the particular case of X. albilineans, it is known that its pathogenicity is directly related to the production of a secondary metabolite, termed albicidin, which shows unique structural features and a remarkable bioactivity against Gram-positive and Gramnegative bacteria.1 The agent for mango bacterial black spot, Xanthomonas citri pv. mangiferaeindicae, is endemic in mangoproducing countries of the world, including Asia, as well as Southern and Eastern Africa.2 In metabolomic studies, different dereplication approaches facilitate structure elucidation of metabolites as well as the investigation of biosynthetic pathways.3 In 2013, Yang et al. presented a dereplication strategy for the discovery of natural products utilizing MSn spectra combined with molecular networking.4 With high-resolution MS instrumentation as a basis, the exploitation of information-dependent acquisition and stable-isotope-labeled precursor feeding have become a powerful tool not only for quantification of proteins, making use of stable isotope labeling of amino acids in cell culture (SILAC) experiments,5 but also to unravel biochemical pathways of secondary metabolites.6,7 Terpenoids, predominantly produced by bacteria and plants, are known for having various ecological functions such as repellents to pests, as well as defense against pathogenic bacteria and fungi. Within the class of terpenoids, there is a high degree of structural diversity and they exhibit a wide range of bioactivities, for example, chemotherapeutic © XXXX American Chemical Society and American Society of Pharmacognosy

(paclitaxel, squalamine) and anti-inflammatory (linalool, 1,8cineole) agents, as well as antiparasitic (artemisinin), antifungal (candicidin D), and antibacterial agents (farnisol).8,9 The attachment of a terpene moiety, derived from the mevalonate or nonmevalonate biosynthetic route, to another structural unit from a different biosynthetic route yields hybrid natural products which have been termed meroterpenoids. Examples are the marine-derived cytotoxic erythrolic acids,10 consisting of a p-hydroxybenzoic acid building block attached to the terpenoid structure. Recent findings of antibacterial oligopeptides1 containing the unusual para-aminobenzoic acid (pABA) have encouraged us to search for new pABA-containing metabolites. This precursor is rarely to be found in secondary metabolites and is mainly utilized in folic acid metabolism.8 In this work, we used a combined approach of isotope-labeled culture feeding together with spectral networking in order to identify secondary metabolites harboring pABA units produced by Xanthomonas citri pv. mangiferaeindicae.

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RESULTS AND DISCUSSION A new meroterpenoid consisting of an isoprenylated pABA unit was identified by differential isotope labeling employing unlabeled pABA and deuterium-labeled-pABA (d4-pABA) and Received: November 24, 2015

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DOI: 10.1021/acs.jnatprod.5b01049 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

purified as a yellow viscous oil. A predominant peak of m/z 468.3098 = [M + H]+ (C29H41O4N Δm 1.46 ppm) was identified as the monoisotopic mass and could be distinguished by a mass shift of 3 Da to lower masses from the d4-pABA-fed culture (m/z 471.3295 = [M + H]+) (Figure 1). This observation suggested an additional substitution at the pABA moiety. MS/MS experiments using collision-induced dissociation with a 30% normalized collision energy (CID-30) resulted in the same fragmentation pattern seen in the MS spectrum and revealed no additional information about the molecular fragments. Various NMR experiments confirmed the pABA spin system as well as the expected substituent in an ortho arrangement to the amino group (chemical shifts are provided in Table 1; see also Figure S2 for NMR data showing this

subsequent analysis of culture broths using LC-ESI-MS/MS analysis. For this, we synthesized d4-pABA and cultured X. citri pv. mangiferaeindicae in media containing pABA and d4-pABA, respectively (details of the synthesis of the d4-pABA is provided in the Supporting Information). Spectral networking was utilized to cluster related MS/MS spectra thereby revealing a pABA-containing metabolite (Figure 1 and Figure S1). In order

Table 1. NMR Spectroscopic Data (700 MHz, C2D6OS) of Xanthomonic Acid

Figure 1. Spectral networking analysis of feeding experiments. (A) MS1 spectra of the target metabolite show a mass difference of 3 Da between pABA (blue) and d4-pABA feeding experiments (red), indicating a monosubstituted pABA moiety to be present in the structure. (B) The corresponding cluster from the network analysis of culture extracts (see Supporting Information for the entire network). (C) MS/MS spectra from the natural (left) and isotope-labeled terpene (right). The mass shifts of 3 Da indicates that all fragments contain the pABA moiety.

a

position

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 NH2 (4) OH (7) OH (25)

123.3, C 130.5, CH 119.5, C 150.5, C 113.6, CH 129.1, CH 168.7, C 29.1, CH2 121.9, CH 136.5, C 39.8, CH2 26.7, CH2 124.4, CH 134.8, C 39.5, CH2 26.3, CH2 124.4, CH 134.6, C 39.6, CH2 26.5, CH2 124.1, CH 134.4, C 35.2, CH2 34.2, CH2 175.4, C 16.4, CH3 16.2, CH3 16.3, CH3 16.3, CH3

δH (J in Hz, integral)

HMBC

7.52, s (0.9)

4, 6, 7, 8

6.59, d (8.2, 1.0)a 7.50, dd (8.2, 1.0)

1, 3 2, 4, 7

3.10, d (7.2, 2.0) 5.30, t (7.1, 1.3)

1, 2, 4, 9, 10 8, 11, 26

2.04, m (n.d., 13.6)b 2.09, m (n.d., 13.6)b 5.12, t (6.9, 1.3)

9, 12, 26 11, 13, 14 12, 15, 27

1.94, t (7.3, 2.7) 2.01, m (n.d., 13.6)b 5.06, m (n.d., 2.4)b

13, 16, 27 15, 17, 18 16, 19, 28

1.90, t (7.4, 2.4) 2.00, m (n.d., 13.6)b 5.07, m (n.d., 2.4)b

17, 20, 28 19, 21, 22 20, 23, 29

2.14 m (n.d., 13.6)b 2.21 m (n.d., 2.3)

21, 25, 29 23, 25

1.66, 1.56, 1.54, 1.54, n.d. n.d. n.d.

9, 10, 11 13, 14, 15 17, 18, 19 21, 22, 23

s s s s

(3.6) (4.0) (6.6)b (6.6)b

Reference signal for 1H integrals. bSignal overlap, n.d. not determined

attachment). The NMR spectra revealed a partially overlapped aliphatic region with a repetitive pattern: the chemical shift region with δ1H 5.0−5.3 ppm suggests there are four protons attached to sp2 carbons (δ13C 121.9−124.4 ppm), a second region (δ 1H 1.9−3.1 ppm and δ 13C 26.3−39.8 ppm) corresponds to nine methylene groups neighboring the aforementioned sp2 carbons, and finally four methyl groups were found in the region of δ1H 1.5−1.6 ppm and δ13C 16.2− 16.4 ppm. The 2D 1H−13C HMBC spectrum showed correlations for two of the methylene protons (δ1H 2.1−2.2

to increase the amount of MS/MS spectra toward lessabundant compounds, we initially fractionated the crude extracts by solid-phase extraction (SPE) and acquired the data in two LC-MS/MS experiments per sample. In the two LC-MS/MS experiments, we adjusted the precursor selection range in the first experiments from m/z 200−600 and in the second from m/z 600−3000. Feeding pABA (3.6 mM) divided in daily doses over 4 days increased the production of a terpenoid in the culture medium. The substance (1 mg) was B



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ppm) to a carboxylic carbon with a chemical shift of δ13C 175.4 ppm (See Figures S3−7 for NMR spectra). The ROE signal patterns between the vinylic protons and the neighboring methylene and methyl groups supported E-configurations for the isolated double bonds (Figure 2B). In summary, the

Figure 2. Structure elucidation. (A) Structure of xanthomonic acid produced by Xanthomonas citri pv. mangiferaeindicae. (B) NMR spectroscopic through-bond (COSY, HMBC) and through-space (ROESY) correlations are illustrated. For simplicity, correlations from the repetitive units of the diterpene segment are shown only twice for both 1H−13C HMBC and 1H−1H ROESY correlations.

analytical data showed that the purified compound, which we named xanthomonic acid, consists of a diterpene element attached to the 3-position of pABA (Figure 2). Interestingly, we could not detect any antibacterial or antifungal activity for xanthomonic acid. We therefore evaluated the biological activity of xanthomonic acid using a modified 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assay for determination of cytotoxicity.11 Xanthomonic acid showed inhibitory effects toward nine human cancerous cell lines plus the noncancerous HEK293 cell line. The molecule shows higher potency against embryonic kidney, cervical, and breast cancer cell lines, with higher selectivity toward estrogen-independent breast cancer cells (MDA-MB-231) compared to the estrogen-dependent type (MCF-7). Reduced activity was observed against brain and prostate cell lines, while activity against colorectal, lung, pancreatic, and bladder cell lines was moderate (Figure 3A,B and Table 2; see also Figure S8 for all cell lines). Following 48 h treatment of cells with xanthomonic acid, vacuole formation was observed to be higher in 253JBV cells compared to both MDA-MB-231 and HeLa cells (Figure 3B). To distinguish between autophagy and apoptosis regarding the cell death mechanism, protein levels were determined after 24 h treatment with xanthomonic acid. Autophagy and the ubiquitin proteasome system (UPS) are two processes for cellular protein degradation. Therefore, an additional immunoblotting assay was performed for analysis of autophagy markers and total polyubiquitinated protein levels for the aforementioned cell lines (Figure 3C). Autophagy plays an essential role in maintaining cellular homeostasis and continuity, as well as for the elimination of pathogens, macromolecules, and organelles, through which it serves as a protective response toward several cellular disorders including tumorigenesis.12 Therefore, agents which induce autophagy can be utilized for concurrent use in cancer suppression.13 On the contrary, other types of cancer which develop autophagy as a protective mechanism require the use of

Figure 3. Bioactivity of xanthomoonic acid (A) Cytotoxic effects of xanthomonic acid were determined by MTT assay as a measure of cell viability following 48 h exposure to different concentrations of xanthomonic acid (0.2, 2, and 20 μg/mL). Controls were exposed to vehicle only, which was taken as 100% viability. Data are expressed as mean ± SD. (B) Microscopic examination of the cancerous 253J-BV (bladder) cells after growth for 48 h without treatment (left) and after treatment with 20 μg/mL xanthomonic acid (right) showing disrupted cells. (C) Immunoblotting assay was performed for analysis of autophagy markers (the conversion of LC3-I to LC3-II, and p62 protein level) and total polyubiquitinated protein levels. MDA-MB231, HeLa, and 253J-BV cell lines were treated with xanthomonic acid at concentrations of IC50 and 2× IC50 for 24 h. Cells were treated with Mg132 (4.76 μg/mL) and BafA1 (0.1 μg/mL) as positive controls and DMSO as negative controls.

autophagy-inhibitors concurrently with chemo- or radiotherapy. Xanthomonic acid significantly increased the processing of the microtubule-associated protein light chain (LC3-I) to the LC3II form which reflects maturation of autophagosomes carrying cellular components to be degraded.12 The increase of the LC3II/LC3-I ratio might be the result of both induction or inhibition of autophagy; therefore, we analyzed the levels of the p62 protein, which is involved in promoting turnover of protein aggregates following poly ubiquitination. The levels of p62 reflect the stream of the autophagic process, where elevated levels suggest defective or reduced autophagic activity. We noticed that xanthomonic acid caused a significant decrease in p62 levels on all analyzed cell lines at twice the half maximal C



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GmbH Co. KG (Karlsruhe, Germany), Merck (Darmstadt, Germany), Sigma-Aldrich (Taufkirchen, Germany), and TCI (Zwijndrecht, Belgium) and if not specified, they were used for the synthesis and analyses without further purification. Deuterated solvents, used for NMR-spectroscopy (chloroform-d1 99.8% and dimethyl sulfoxide-d6 99.8%), were purchased from Deutero GmbH (Kastellaun, Germany). Thin-layer chromatography (TLC) was performed using TLC plates, purchased from Merck (Silica gel 60, F254, coating thickness 0.2 mm). The compounds were detected by UV-light with wavelength λ = 254 nm or staining with Ninhydrin solution. Flash chromatography was performed with silica gel from Merck and Machery & Nagel (Düren, Germany) (particle size 0.04−0.063 mm). Automatic flash chromatography was performed on a CombiFlash Rf 200 system, with two channel diode array detector (wavelength range λ = 200−360 nm), combined with RediSep Rf RP C18 columns (Teledyne ISCO, Lincoln, NE, U.S.A.). 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 and Bruker Avance 500 NMR-spectrometer (Bruker, Rheinstetten, Germany). The residual protonated solvent signals were used as standards. Chemical shifts are given in δ-units (ppm) relative to the solvent signal. High-resolution mass-spectrometry (HRMS) using the ESI-technique was performed on a LTQ Orbitrap XL, produced by Thermo Scientific (Waltham, MA, U.S.A.). IR spectra were recorded on a Jasco FT-IR 4100 spectrometer (Jasco, Groß Umstadt, Germany). NMR spectra of xanthomonic acid were acquired on an Avance III 700 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) equipped with a TXI 1.7 mm room temperature probe head. A sample of 1 mg was dissolved in 45 μL d6-dimethyl sulfoxide (DMSO) and filled into a 1.7 mm NMR tube. All experiments were performed at 298 K. Chemical shifts were referenced relative to tetramethylsilane (TMS; 1H, 13C) respectively. All NMR experiments used standard parameters sets employed by the manufacturer (Bruker, Karlsruhe, Germany). For the bioactivity studies, the antibodies SQSTM1/p62 (5114) and LC3A/B (4108) were purchased from Cell Signaling Technology (Danvers, Massachusetts, U.S.A). Polyubiquitin antibody (sc-8017) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, U.S.A). Actin antibody (A2228), Mg132 and BafilomycinA1 were purchased from SigmaAldrich (Taufkirchen, Germany). Synthesis of d4-pABA. See Supporting Information. Fermentation of Xanthomonas citri pv. mangiferaeindicae. The strain was obtained from CIRM-Plant Associated Bacteria (CIRM-CFBP, Beaucouzé Cedex, France). Liquid cultures of the wild type producer Xanthomonas citri pv. mangiferaeindicae (strain CFBP 7237) were grown at 28 °C and agitated at a speed of 160 rpm on a rotary shaker (Infors HT Multitron Standard, Bottmingen, Switzerland). Several batches of 1 L volume were grown in YPG medium (glucose 7.0 g/L; bacto peptone 7.0 g/L; yeast extract 7.0 g/ L; pH 7.3) for 5 days. After the first 24 h of growth, the cultures were fed (125 mg/L culture) pABA daily for 4 days. pABA was dissolved in DMSO (ROTH, Karlsruhe, Germany) and sterile filtered (0.22 μm). Although the target compound could be detected through LC-MS analysis, an increase in metabolite production could be observed with this feeding procedure. Isolation and Purification. Cultures were centrifuged at 4000 rpm for 1 h to obtain a clear aqueous supernatant (1 L), which was extracted using XAD-1600 Amberlite (10% w/v) for 16 h. The target compound was eluted from the XAD material using MeOH/H2O stepwise (10% MeOH, followed by 90% MeOH). The 90% MeOH eluate was dried in vacuo to yield a reddish-brown extract (200 mg). Solid-phase extraction on a C8 CHROMOBAND column (10 g) reduced the amount of impurities in the sample, with the target compound eluted using 60% acetonitrile/H2O (10 CV). The organic solvent was removed in vacuo using a rotary evaporator, followed by removal of water by freeze-drying. The dried extract (5.5 mg) was purified by preparative HPLC on an Agilent 1100 system (Agilent, Waldbronn, Germany) at detection wavelengths of λ = 280 nm on a C18 reversed-phase column (GromSil 120 ODS-5 ST, 10 μm Length: 250 mm ID: 20 mm, Grace, Rottenburg-Hailfingen, Germany) using a linear acetonitrile gradient starting from 30% to 90% acetonitrile (containing 0.1% HCOOH) over 40 min with a flow rate of 15.0 mL/

Table 2. IC50 Values (μM) for Xanthomonic Acid and the Positive Control (Parthenolide) against Different Cell Lines Following 48 h Exposure cell line

xanthomonic acid (μM)

parthenolide (μM)

HeLa (cervix) CaCo-2 (colorectum) MDA-MB-231 (breast) MCF-7 (breast) U87MG (brain) HEK293 (kidney) A549 (lung) mPanc-96 (pancreas) PC3 (prostate) 253J-BV (bladder)

18.24 ± 0.47 25.91 ± 0.90 12.87 ± 0.04 >42.83 >42.83 6.45 ± 0.03 25.76 ± 0.13 36.42 ± 1.35 42.31 ± 0.90 36.51 ± 1.00

23.08 ± 0.24 23.40 ± 0.81 4.36 ± 0.04 27.71 ± 1.53 4.86 ± 0.10 3.10 ± 0.12 22.03 ± 2.10 20.30 ± 0.44 13.01 ± 0.56 25.29 ± 1.17

inhibitory concentration (IC50), suggesting induction of autophagy rather than inhibition. Treatment of HeLA cells with xanthomonic acid at IC50 also showed a significant decrease of p62 levels. Finally, we assessed the levels of total polyubiquitinated proteins, where elevated levels reflect low turnover and thus reduced autophagic activity. The level of polyubiquitinated proteins decreased for MDA-MB-231 cells at both doses (IC50 and twice the IC50 concentrations), and the decrease for 253J-BV cells was only observed at the IC50 concentration (Figure 3C). Comparisons were made in reference to the proteasome inhibitor MG132 (4.76 μg/mL) and bafilomycin A1 (BafA1, 0.1 μg/mL) for positive controls, and to DMSO as the negative control. BafA1 is an autophagy inhibitor which inhibits lysosomal degradation by decreasing acidification of lysosomes by inhibiting vacuolar type H+ ATPase (V-ATPase);14 while Mg132 is a proteasome inhibitor which acts by reducing the degradation of ubiquitin-conjugated proteins in mammalian cells.15 These findings showed that xanthomonic acid induced cell death via the activation of autophagy in 253J-BV, MDA-MB-231, and HeLa cells. The activation of autophagy by xanthomonic acid was shown to be higher for the 253J-BV cells than for both HeLa and MDA-MB231 cells. High activation against the former was observed at IC50 concentrations, whereas activation was moderate at 2× IC50 for the latter two cell lines, which is an attribute that favors bladder tumor cells for potential therapeutic targets in future studies. Xanthomonic acid follows earlier reports of terpene metabolites from the genus Xanthomonas.16 We could detect this meroterpenoid by LC-MS without feeding pABA, yet in much lower abundance. By contrast, feeding para-hydroxybenzoic acid did not result in isoprenylation in a similar manner, suggesting specificity of the biosynthetic enzyme ligating pABA and the terpenoid element. Moreover, the spectral network analysis revealed other potential candidates for pABA-containing metabolites. Future investigations may identify these structures and clarify whether they originate from primary or secondary metabolism. Our results also bring attention to structural attributes of meroterpenoids as cytotoxic lead molecules of relatively small size. Understanding the underlying mechanism of action (i.e., stimulation of autophagy) is important for utilization as a tumor suppressor.

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EXPERIMENTAL SECTION

General Experimental Procedures. All chemicals were obtained from commercial suppliers such as ABCR (Karlsruhe, Germany), Acros (Geel, Belgium), Alfar Aesar (Karlsruhe, Germany), Carl Roth D



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Determination of Half Minimal Inhibitory Concentration (IC50). Cytotoxicity was expressed as mean percentage increase relative to the unexposed control ± SD. Control values was set to 0% cytotoxicity. Cytotoxicity data (where appropriate) were fitted to a sigmoidal curve, and a four-parameter logistic model was used to calculate the IC50, which is the concentration causing 50% inhibition in comparison to untreated controls. The mean IC50 is the concentration of agent that reduces cell growth by 50% under the experimental conditions and is the average from at least three independent measurements that will be reproducible and statistically significant. The IC50 values were reported at ±95% confidence intervals (±95% CI). This analysis was performed with Graph Pad Prism 5 (San Diego, CA, U.S.A.). Morphological Studies. The morphological studies of the cells were performed with an inverted microscope (Olympus, Tokyo, Japan) compared to the control group 48 h after treatment (see Supporting Information: SI Figure 2). Immunoblotting Assay. Autophagy markers and total polyubiquitinated protein levels from MDA-MB-231, HeLA, and 253J-BV cell lines were analyzed following treatment with xanthomonic acid at concentrations of 1/2 IC50, IC50 and 2× IC50 for 48 h. Mg132 (4.76 μg/mL) and BafA1 (0.1 μg/mL) were used as positive control agents. Cells were seeded at 2.5 × 105 in 6-well plates and 24 h later treated with xanthomonic acid. Treated and untreated cells were harvested. Cells were lysed with RIPA lysis buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 1% protease inhibitor cocktail (M250, Amresco, Ohio, U.S.A.). The resulting suspension was then centrifuged at 14 000 rpm at 4 °C for 10 min, and supernatant was collected. The concentration of protein was determined using the BCA assay (Thermo Fisher Scientific, Bremen, Germany, 23225). Protein extracts (30 μg) were loaded on 15% Tris/ glycine gels for SDS-PAGE, and then proteins were transferred onto Immobilon-P membranes (Millipore Corp., Darmstadt, Germany, IPVH00010). After blocking with 5% skim milk for 30 min, the membrane was incubated 1 h at room temperature with the primary antibody and subsequently washed 30 min and incubated with a secondary horseradish peroxidase (HRP) conjugated antibody (Thermo Fisher Scientific, Bremen, Germany, 32430, 31460). Protein bands were detected using ECL substrate (Bio-Rad, California, U.S.A., 1705061) and monitored with Vilber Lourmat Fusion Fx-7 image and analytics system.

min, followed by an isocratic step (100% acetonitrile) for another 5 min. The target compound was eluted at a retention time Rt = 30.9 min in yields of 0.33 mg/L culture. HPLC-ESI-MS/MS. MS and MS/MS measurements were performed on an LTQ-Orbitrap XL hybrid instrument (Thermo Fisher Scientific GmbH, Bremen, Germany) coupled to analytical HPLC 1200 system (Agilent, Waldbronn, Germany) over a C18 Core−shell column (Supelco Ascentis Express 10.0 × 2.1 mm, 10 μm, SigmaAldrich, St. Louis, MO, U.S.A.), with a linear gradient from 5% to 100% acetonitrile plus 0.1% HCOOH over 20 min followed by 5 min washout and 5 min re-equilibration, with a flow rate of 0.4 mL/min. MS1 survey scans were performed in the Orbitrap analyzer with R = 15 000 at m/z 400 and maximum filling time of 200 ms. LC-MS/MS experiments of the three most intense ions were performed in the LTQ using CID (30 ms activation time); the collision energy was set to 30%. Precursor-ion isolation was performed within a range of m/z 200−600 in a first LC-MS/MS run and in the second from m/z 600− 3000. Masses with unresolved charged state and charge states z > 4 were excluded. Dynamic exclusion was set up for a m/z 2.0 windows for up to 50 precursor ions with a repeat of 2 within 30 s. Samples were analyzed in two LC-MS/MS runs. In the first run, the precursor mass selection window was set to m/z 200−600 and in the second run to m/z 600−3000. MS/MS Network Analysis. MS data sets were converted to mzXML using MSconvert of the Proteowizard software packet.17 After file conversation, the data sets were uploaded for network analysis to gnps (gnps.ucsd.edu). The data sets were divided into two groups (G1, light and G2, heavy). Precursor mass tolerance was set to m/z 0.1 and MS/MS tolerance to m/z 0.5. Cosine cutoff was set to 0.7, Network TopK to 10, the maximum compound connections to 100 with minimum 3 matching peaks and a minimum cluster size of two MS/ MS spectra. The network was then visualized in Cytoscape18 with a force directed layout based on the cosine value. Cell Lines and Maintenance. HeLa (human cervix adenocarcinoma), A-549 (human alveolar adenocarcinoma), MCF-7 (human estrogen-dependent breast adenocarcinoma), MDA-MB-231 (human estrogen-independent breast adenocarcinoma), CaCo-2 (human colon colorectal adenocarcinoma), mPANC96 (human pancreas adenocarcinoma), PC-3 (human prostate adenocarcinoma), 253J-BV (human bladder cancer cells), U87MG (human glioblastoma-astrocytoma), and a normal cell line HEK293 (human embryonic kidney cells) were used for testing cytotoxicity. All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, U.S.A.) except for 253J-BV cells, which were obtained from Creative Bioarray (Shirley, NY, U.S.A.). The cell lines were maintained in Dulbecco’s modified Eagle’s medium F12 (DMEM/F12), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Lonza, Visp, Switzerland). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells were subcultured twice a week, and cells in the exponential growth phase were used in the experiments. Cytotoxicity Assay. Cytotoxicity of xanthomonic acid was determined using a modified MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide)] assay, which detects the activity of mitochondrial reductase of viable cells.7 The assay principle is based on the cleavage of MTT that forms formazan crystals by cellular succinate-dehydrogenases in viable cells. The addition of DMSO to wells helps formazan crystals to be dissolved. For this purpose, all cell lines were cultivated for 24 h in 96-well microplates with an initial concentration of 1 × 104 cells/well in a humidified atmosphere with 5% CO2, at 37 °C. Then, the cultured cells were treated with different dilutions of xanthomonic acid (0.2, 2, 20 μg/mL) followed by incubation for 48 h at 37 °C. Parthenolide (Sigma, St. Lois, MO, U.S.A.) was used as a positive control. The optical density of the dissolved material was measured at λ = 570 nm (reference filter, λ = 620 nm) with UV−vis spectrophotometer (Thermo Multiskan Spectrum). The viability (%) was determined by the following formula: %viable cells = [(absorbance of treated cells) − (absorbance of blank)]/[(absorbance of control) − (absorbance of blank)] × 100.

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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.5b01049. Detailed synthesis of d4-pABA; supporting figures for spectral network analysis; NMR and bioactivity studies (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Sebastian Kemper (Technical University of Berlin) for the instrumentation of the NMR experiments and Dr. P. Ballar Kırmızıbayrak (Ege University) for support and guidance on the autophagy study. H.S. thanks the collaborative funding and support from the German Academic Exchange Service (Deutscher Akademischer Austauschdienst) and the Egyptian Ministry of Higher Education. E



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