New Cytotoxic Cyclic Peptide from the Marine Sponge-Associated Nocardiopsis sp. UR67

A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds—tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)—were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including 1D and 2D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey’s method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.


Introduction
Actinomycetes are a diverse group of aerobic Gram-positive microorganisms withhigh guanine-cytosine DNA content [1]. They belong to the phylum Actinobacteria, which is one of the largest bacterial phyla, distributed in both terrestrial and marine ecosystems [2,3]. About 70% of all naturally derived drugs in clinical use originate from Actinobacteria as they contain biologically active secondary metabolites accounting for their clinical use, mainly as antibacterial, antifungal,

Results and Discussion
Nocardiopsis sp. UR67 was cultivated from the sponge Callyspongia sp. (family Callyspongiidae) that was collected from the Red Sea (Ras Mohamed, Sinai, Egypt; (GPS: 27 • 47.655 N; 34 • 12.904 W) in August 2008. ISP2 liquid broth with calcium alginate beads [27] of Nocardiopsis sp. UR67 was extracted with ethyl acetate, and the obtained organic extract was fractionated on Sephadex LH20. This was followed by purification using semi-preparative reversed phase high performance liquid chromatography (HPLC) to yield a new cyclic hexapeptide nocardiotide A (1), along with three known compounds-tryptophan (2), kynurenic acid (3), which was isolated for the first time from microbial origins, and 4-amino-3-methoxy benzoic acid (4) (Figure 1).
Mar. Drugs 2018, 16, x FOR PEER REVIEW 2 of 13 belonging to the family Nocardiopsaceae and as morphologically similar to members of the genera Actinomadura and Nocardia [7,8]. By reviewing the literature on the genus Nocardiopsis [9,10], it has been clearly demonstrated that it is a prolific producer of a wide variety of bioactive compounds, mainly cyclic peptides [11,12], polyketides [13,14], macrolides [15], alkaloids [16], diketopiperazines [17,18], α and γ-pyrones [19,20], naphthoquinones [21], phenazines [22], and phenoxazine derivatives [23], which contributes to a broad spectrum of biological activities, mainly as cytotoxic [21], anticancer [22], antitumor [24], antibacterial [11], antifungal [25], immunemodulatory [15],and protein kinase inhibitory [26]. Cancer still remains one of the most serious challenges to human health. Despite intense efforts to develop treatments, effective-particularly highly selective-agents are still not available for many cancer types. Therefore, it is necessary to continue the discovery of new classes of molecules with cytotoxic activity. One strategy to treat cancer is to find compounds with new scaffolds that have increased chances of possessing novel binding modes or even addressing novel targets. Consequently, this current investigation is a continuation of our efforts to seek new, effective cytotoxic agents from actinomycetes-associated with marine sponges, specifically, the Nocardiopsis sp. UR67 strain-and to evaluate their cytotoxic biological activities.

Results and Discussion
Nocardiopsis sp. UR67 was cultivated from the sponge Callyspongia sp. (family Callyspongiidae) that was collected from the Red Sea (Ras Mohamed, Sinai, Egypt; (GPS: 27°47.655′ N; 34°12.904′ W) in August 2008. ISP2 liquid broth with calcium alginate beads [27] of Nocardiopsis sp. UR67 was extracted with ethyl acetate, and the obtained organic extract was fractionated on Sephadex LH20. This was followed by purification using semi-preparative reversed phase high performance liquid chromatography (HPLC) to yield a new cyclic hexapeptide nocardiotide A (1), along with three known compounds-tryptophan (2), kynurenic acid (3), which was isolated for the first time from microbial origins, and 4-amino-3-methoxy benzoic acid (4) (Figure 1).
These six amino acids accounted for18 degrees of unsaturation, indicating that nocardiotide A (1) was a monocyclic hexapeptide. The absolute configurations of the amino acid units in nocardiotide A (1) were determined by acid hydrolysis, followed by chiral derivatization with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-lalanine amide, FDAA). HPLC analysis of the Marfey's derivatives in comparison to their respective D-and L-authentic reference amino acids revealed the absolute configuration of all amino acids of the new cyclic hexapeptideto beL.

Biological Activities of the Isolated Compounds
The four aforementioned isolated compounds were examined for their cytotoxicity potential towards the murine CT26 colon carcinoma, the human HeLa cervix carcinoma, and the human MM.1S multiple myeloma cell lines. Nocartiodite A (1) displayedprominent cytotoxic features with IC 50 values of 8, 11, and 12 µM/mL against the human MM. 1S multiple myeloma, human HeLa cervix carcinoma, and murine CT26 colon carcinoma, respectively ( Figure 4). Tryptophan (2), kynurenic acid (3), and4-amino-3-methoxy benzoic acid (4) did not demonstrate any considerable cell death properties at the examined concentration.

Biological Activities of the Isolated Compounds
The four aforementioned isolated compounds were examined for their cytotoxicity potential towards the murine CT26 colon carcinoma, the human HeLa cervix carcinoma, and the human MM.1S multiple myeloma cell lines. Nocartiodite A (1) displayedprominent cytotoxic features with IC50 values of 8, 11, and 12 μM/mL against the human MM. 1S multiple myeloma, human HeLa cervix carcinoma, and murine CT26 colon carcinoma, respectively ( Figure 4). Tryptophan (2), kynurenic acid (3), and4-amino-3-methoxy benzoic acid (4) did not demonstrate any considerable cell death properties at the examined concentration.

General Experimental Procedures
Melting points were measured using Stuart Scientific (SMPI) melting point apparatus and were uncorrected. An ultraviolet lamp (CAMAG, Wilmington, NC, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm. 1 H (600 MHz) and 13 C (150 MHz) NMR spectra were recorded on BrukerAvance III HD 600 instruments (Bruker Biospin, Rheinstetten, Germany) in CD3OD. The samples were degassed by an ultrasonic water bath (Branson 3800 Ultrasonic Cleaner, Branson, Gayton, UK) for 20 min before measurements. Solvent signals of CD3OD (δH 3.3 ppm and δC 49.0 ppm) were considered as the internal reference signals for calibration. Chemical shift values (δ) were recorded in ppm units and coupling constants (J) in Hz. Heteronuclear correlations were measured using HSQC (optimized for 1 JHC = 145 Hz) and HMBC (optimized for n JHC = 8.3 Hz or n JHC = 4.0 Hz) pulse sequences. Positive and negative HR-ESI-MS spectra were obtained using a Synapt G2 HDMS QTOF-mass spectrometer (Waters, Eschborn, Germany). HPLC separations and purifications were performed on the Knauer system (Knauer, Berlin, Germany). This included Smartline S-1000 quaternary pumps coupled with a Smartline S-2600 UV-VIS multiwavelength detector (Knauer, Berlin, Germany), a Knauer dynamic mixing chamber, and using a C18 column (5 μm, 10 mm × 250 mm, Knauer, Berlin, Germany) at ambient temperature with a guard column filled with the same stationary phase. On the other hand, the analytical detection was carried out using an analytical Gemini-NX RP-18 column (5 μm, 4.60 mm × 100 mm; Phenomenex, Aschaffenburg, Germany).

Isolation, Fermentation,and Extract Preparation of Nocardiopsis sp. UR67
Sponge specimens were rinsed in sterile seawater, cut into pieces of ca. 1 cm 3 , and then thoroughly homogenized in a sterile mortar with 10 volumes of sterile seawater. The supernatant was diluted in a tenfold series (10 −1 , 10 −2 , 10 −3 ) and subsequently plated out on agar plates. Four different media-M1, ISP medium 2, Oligotrophic medium (OLIGO), and Marine Agar (MA)-were

General Experimental Procedures
Melting points were measured using Stuart Scientific (SMPI) melting point apparatus and were uncorrected. An ultraviolet lamp (CAMAG, Wilmington, NC, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm. 1 H (600 MHz) and 13 C (150 MHz) NMR spectra were recorded on BrukerAvance III HD 600 instruments (Bruker Biospin, Rheinstetten, Germany) in CD 3 OD. The samples were degassed by an ultrasonic water bath (Branson 3800 Ultrasonic Cleaner, Branson, Gayton, UK) for 20 min before measurements. Solvent signals of CD 3 OD (δ H 3.3 ppm and δ C 49.0 ppm) were considered as the internal reference signals for calibration. Chemical shift values (δ) were recorded in ppm units and coupling constants (J) in Hz. Heteronuclear correlations were measured using HSQC (optimized for 1 J HC = 145 Hz) and HMBC (optimized for n J HC = 8.3 Hz or n J HC = 4.0 Hz) pulse sequences. Positive and negative HR-ESI-MS spectra were obtained using a Synapt G2 HDMS QTOF-mass spectrometer (Waters, Eschborn, Germany). HPLC separations and purifications were performed on the Knauer system (Knauer, Berlin, Germany). This included Smartline S-1000 quaternary pumps coupled with a Smartline S-2600 UV-VIS multiwavelength detector (Knauer, Berlin, Germany), a Knauer dynamic mixing chamber, and using a C18 column (5 µm, 10 mm × 250 mm, Knauer, Berlin, Germany) at ambient temperature with a guard column filled with the same stationary phase. On the other hand, the analytical detection was carried out using an analytical Gemini-NX RP-18 column (5 µm, 4.60 mm × 100 mm; Phenomenex, Aschaffenburg, Germany).

Isolation, Fermentation, and Extract Preparation of Nocardiopsis sp. UR67
Sponge specimens were rinsed in sterile seawater, cut into pieces of ca. 1 cm 3 , and then thoroughly homogenized in a sterile mortar with 10 volumes of sterile seawater. The supernatant was diluted in a tenfold series (10 −1 , 10 −2 , 10 −3 ) and subsequently plated out on agar plates. Four different media-M1, ISP medium 2, Oligotrophic medium (OLIGO), and Marine Agar (MA)-were used for the isolation of actinobacteria. All media were supplemented with 0.2 µm pore size filtered cycloheximide (100 µg/mL), nystatin (25 µg/mL), and nalidixic acid (25 µg/mL) to facilitate the isolation of slow-growing actinobacteria; cycloheximide and nystatininhibit fungal growth, while nalidixic acid inhibits many fast-growing Gram-negative bacteria [44]. All media contained DifcoBacto agar (18 g/L) and were prepared in 1 L artificial sea water (NaCl 234.7 g, MgCl 2 ·6 H 2 O 106.4 g, Na 2 SO 4 39.2 g, CaCl 2 11.0 g, NaHCO 3 1.92 g, KCl 6.64 g, KBr 0.96 g, H 3 BO 3 0.26 g, SrCl 2 0.24 g, NaF 0.03 g, and ddH 2 O to 10.0 L). The inoculated plates were incubated at 30 • C for 6-8 weeks. Distinct colony morphotypes were picked and restreaked until visually free of contaminants. The isolates were maintained on plates for short-term storage and long-term strain collections. Nocardiopsis sp. UR67 was fermented in 10 Erlenmeyer flasks (2 L), each containing 1 L of ISP 2 (International Streptomyces Project) medium in artificial sea water and incubated at 30 • C for 10 days with shaking at 150 rpm. After fermentation and filtration, the supernatant was extracted with ethyl acetate (3 × 500 mL) to give the organic extract for subsequent compound isolation.

LC-HR/MS Analysis
Ethyl acetate extract of 1 mg/mL in MeOH was analyzed on an Accela HPLC (Thermo Scientific, Karlsruhe, Germany) coupled to a UV detector at 280 and 360 nm and an Exactive-Orbitrap high resolution mass spectrometer (Thermo Fisher Scientific, Karlsruhe, Germany). The HPLC column was an ACE (ACE, Mainz, Germany) C18, 75 mm × 3.0 mm, 5 µm column. The mobile phase consisted of purified water (A) and acetonitrile (B) with 0.1% formic acid in each solvent. The gradient program started with 10% B linearly increased to 100% B at a flow rate of 300 µL/min for 30 min and remained isocratic for 5 min before linearly decreasing back to 10% B in 1 min. The column was then re-equilibrated with 10% B for 9 min before the next injection. The total analysis time for each sample was 45 min. The injection volume was 10 µL, and the tray temperature was maintained at 12 • C. High resolution mass spectrometry was carried out in both positive and negative ESI ionization modes with a spray voltage at 4.5 kV and capillary temperature at 320 • C. The mass range was set from m/z 150-1500. Both negative and positive ionization switch modes were used to include the highest number of metabolites from the investigated bacterial fractions subjected to LC-HR-ESIMS analysis. The dereplication was achieved for each m/z ion peak with metabolites recorded in the customized databases based on established parameters (m/z threshold of ±3 ppm and retention time) [45], which provided a high level of confidence in metabolites identity; consequently, the number of the remaining unknown metabolites in each bacterial fraction was refined.

Marfey's Analysis
The absolute configurations of the amino acids in compound 1 were elucidated by Marfey's derivatization and compared to the corresponding standard amino acids each with D and L configurations (Sigma, Darmstadt, Germany) by HPLC. Compound 1 (1 mg) was initially hydrolyzed with 6 M HCl (2 mL) in a water bath at 100 • C for 24 h. The hydrolysate was cooled to room temperature, dried using a vacuum evaporator and dissolved in 100 µL of water. The Marfey's derivatization was carried out by adding 100 µL of 1% Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amid) dissolved in acetone and 20 µL of 1 M NaHCO 3 (H 2 O) to 50 µL of the hydrolysate of compound 1 as well as 50 mM standard amino acid, respectively, and incubated at 40 • C for 1 h with frequent shaking. The reaction was stopped by adding 10 µL of 2 M HCl after cooling. The Marfey's derivatization products were finally dried and prepared in MeOH for further HPLC analysis. The HPLC chromatography was carried out on Gemini-NX RP-C18 column by eluting with H 2 O/acetonitrile (95:5%) for the first 5 min, linearly gradient to 100% acetonitrile for 30 min, and staying at 100% actonitrile for a further 10 min with a flow rate at 1 mL/min and UV detection at 340 nm. The configuration was eventually determined with the observation of the same retention times compared to the standard enantiomeric amino acids [46][47][48].

Cytotoxic Activity
The cytotoxicity of the isolated compounds was evaluated in cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CT26, HeLa, and MM.1S cells were maintained in RPMI medium (Merck, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS), and grown at 37 • C and 5% CO 2 . HeLa cells (2 × 10 4 per well) were plated in 96-well tissue culture plates in 100 µL cell culture medium. The following day, cells were stimulated overnight in triplicates with the reagents of interest. Cell viability was assessed by crystal violet staining. In case of the CT26 and MM.1S cell lines, cells were seeded in 96-well plates (7× 10 4 cells per well) and were challenged the same day overnight with the reagents of interest; the cytotoxic effect was evaluated using the MTT assay [49]. To normalize cell viability values, each plate included a triplicate of cells treated with the compound carrier DMSO to define 100% viable cells as well as a triplicate of cells incubated with a cytotoxic mixture (200 ng/ML Tumor Necrosis Factor TNF, 200 ng/mL CD95L (Fas ligand), 200 ng/mL TRAIL (TNF-related apoptosis-inducing ligand), 25 µg/mL CHX (Cycloheximide), 1% (w/v) sodium azide) to define maximal cell death and thus 0% viability. All other viability values were normalized according to the averages of these triplicates and analyzed by the Graph Pad Prism 5 software (La Jolla, CA, USA).

Conclusions
In continuation of our interest to isolate and identify new antiproliferative agents from natural sources, the chemical characterization of Nocardiopsis sp.UR67-an actinomycete associated with the sponge (Callyspongia sp.) previously collected from the Red Sea-was conducted alongside with evaluation of the cytotoxic properties of the attained compounds versus the murine CT26 colon carcinoma, the human HeLa cervix carcinoma, and the human MM.1S multiple myeloma cell lines. Ten known metabolites were identified by dereplication using LC-HR-ESI-MS techniques. Additionally, four compounds were isolated and characterized for the first time from the broth culture of Nocardiopsis sp. UR67. Most importantly, one new cyclic hexapeptide-nocardiotide A-was identified, along with tryptophan, kynurenic acid, and 4-amino-3-methoxy benzoic acid. Among them, only nocardiotide A demonstrated significant cytotoxic property.