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Euglenatides, Potent Antiproliferative Cyclic Peptides Isolated from the Freshwater Photosynthetic Microalga Euglena gracilis

Abstract By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non‐proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3 μM in Aspergillus fumigatus and 0.29 μM in MCF‐7 breast cancer cells. In an unprecedented convergence of non‐ribosomal peptide synthetase and polyketide synthase assembly‐line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide‐like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products.


Figure S45
Comparison of CD spectra between euglenatide E and those reported for nemamide A and triene diastereomers. [9] . Materials and methods

General methods and materials
Unless stated otherwise, all chemicals and solvents were purchased from Sigma-Aldrich, Alfa Aesar or Fisher Scientific. All solvents were of HPLC grade or equivalent.
Commercial media and media components were purchased from Sigma-Aldrich, Alfa Aesar, Fisher Scientific or Formedium. Unless stated otherwise, the pH of media was adjusted to 4.8 with 1 N HCl and 1 N KOH prior to autoclaving. UV spectra were acquired on an SPD-M20A photodiode array detector attached to a Shimadzu Prominence/Nexera UHPLC. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for 1 H and 13 C NMR, respectively) equipped with a 1.7-mm TCI MicroCryoProbe. Chemical shifts were reported in ppm using the signals of the residual solvent as internal reference (δH 2.50 and δC 39.5 ppm for DMSO-d6). Optical rotations were acquired on a Jasco P-2000 polarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were measured with a JASCO FT/IR-4100 spectrometer (JASCO Corporation) equipped with a PIKE MIRacle™ (JASCO Corporation) single reflection ATR accessory.

Inocula preparation
Euglena gracilis var. saccharophila Klebs (strain 1224/7A) was obtained from the Culture Collection of Algae and Protozoa (CCAP) (https://www.ccap.ac.uk) and treated with antibiotics to produce axenic cultures using the protocol recommended by CCAP. [1] Cultures were cultivated in EG:JM (Euglena gracilis medium (Table S1) plus Jaworski's Medium (Table S2), replacing ''Lab-Lemco'' with Tryptone) [2] and incubated in a light chamber at 22 o C on a 14 h light /10 h dark cycle with a light intensity of 100 μmol. photons. m -2 s -1 . After four days of incubation, E. gracilis cells were harvested by centrifugation at 2000 x g and 10 o C for 5 min and suspended in EG:JM medium. The absorbance at 740 nm was adjusted on a CLARIOstar microplate reader to 0.1 (≈ 4 g of wet cells per litre) using EG:JM medium as a diluent and blank. Cells were harvested by centrifugation at 2000 x g and 10 o C for 5 min, washed three times with sterile Milli-Q water to remove any trace components of EG:JM medium, and suspended in the medium used for cultivation. This suspension was used to inoculate the production media to obtain approximately 0. After six days, cultures were centrifuged at 3000 x g and 4 o C for 20 min, and cells were vortexed in 10 mL 90% MeOH. Cell debris was removed by centrifugation at 3000 x g and 4 o C for 10 min. After centrifugation, the solvents were evaporated, and the residue of each extract was then dissolved in 1 mL MeOH and passed through SOLA HRP SPE cartridges (Thermo Fisher Scientific) and a 0.2 μm PTFE filter (Whatman, Sigma-Aldrich). For cultivation in the dark, the media were supplemented with 15 g/ L glucose (Glc) and incubated at 30 o C and shaken at 200 rpm in the dark for four days after inoculation with E. gracilis cells.

High resolution MS
For the accurate mass measurement, high-resolution mass spectra were acquired on a Synapt G2-Si mass spectrometer (Waters) operated in negative ion mode with a scan time of 1.0 second in the mass range of m/z 50 to 1200. An aliquot of 7 μL of each sample was injected onto an Acquity UPLC® BEH C18 column, 1.7 µm, 1x100 mm (Waters) maintained at 45 °C and eluted with mobile phases A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) at a flow rate of 80 µL/min. The following gradient was used: 0-1 min (15% B), 11 min (99% B), 11.1 min (15 % B), 15 min (15 % B). The following parameters were used: capillary voltage = 2.5 kV; cone voltage = 40 V; source temperature = 130 °C; desolvation temperature = 350 °C; source offset 80 V. Leu-enkephalin peptide was used as a lock mass (m/z = 554.2620) measured every 30 seconds during the run.

Determination of the optimum fermentation period
A one-litre flask containing 500 mL of synthetic medium + 30 mM Glu was inoculated with Euglena cells to obtain approximately 0.2 g of wet cells per litre (1:20 dilution). An aliquot of 10 mL was taken every day for 13 days, starting 24 h after inoculation, and centrifuged at 3000 x g and 4 o C for 20 min. The cells were harvested and extracted with 90% MeOH. The solvent was evaporated, and the residue of the extract was stored in a freezer at -20 o C until HPLC analysis. Samples were prepared immediately before analysis by adding 1 mL MeOH and passing the solutions through SOLA HRP SPE cartridges (Thermo Fisher Scientific) and a 0.2 μm PTFE filter (Whatman, Sigma-Aldrich).
8. Large-scale cultivation, isolation and purification of euglenatides Synthetic medium + 30 mM Glu (18 L) was inoculated with Euglena cells to obtain approximately 0.2 g of wet cells per litre (1:20 dilution). The culture was cultivated at ambient temperature 22-25 o C under daylight lamps (2000 lumens). After ten days, the culture was centrifuged at 3130 x g and 4 o C for 20 min, and the pellet (53 g) was extracted with 1 L 90% MeOH on a magnetic stirrer. After an hour, the aqueous MeOH extract was partitioned with an equal volume of hexane to remove lipids and pigments.
The aqueous MeOH layer was centrifuged to remove the debris and then evaporated to obtain the residue (1.68 g). This amount was dissolved in 50 mL MeOH along with 15 g of C18 powder, and the solvent was evaporated. The resulting powder was halved into two columns packed with 65 g reversed-phase resin, previously equilibrated with 20% acetonitrile before a frit was added on the top. The extract loaded on the column was fractionated on an automated flash-chromatography system (CombiFlash Rf, Teledyne Isco) using 18 mL/min flow rate and a linear gradient from 20% to 70% acetonitrile in water (in 40 min) followed by a ramp to 100% acetonitrile in 4 min before washing the column for 16 min.
Absorbance was measured at 612 nm with a multimode plate reader (EnVision Perkin Elmer) at T0 (zero time) immediately before incubation at 37 °C for 20 h. After this period, the assay plates were agitated using a DPC Micromix-5, and the absorbance was measured at Tf (final time). Growth inhibition was calculated using the equation mentioned above.
To test the antifungal activity against A. fumigatus, PDA plates were flooded with Tween saline (0.025% v/v of Tween 80 and 8 g/L NaCl), and colonies were harvested in RPMI liquid medium by gently scraping the surface of the agar with a sterile spatula to prepare a conidial suspension. This suspension was filtered through sterile chiffon, and the concentration was determined by counting the conidia in a Neubauer chamber. The inoculum was approximately 2.5 × 10 4 CFU/mL. Resazurin stock solution of 0.02 g in 100 mL was prepared from resazurin sodium salt (R7017, Sigma Aldrich) in Milli-Q water, sterilised by filtration and used as an indicator of eukaryotic cell viability (0.002% final concentration). Resazurin is a blue oxidation-reduction dye that is itself weakly fluorescent until it is irreversibly reduced to the pink-coloured and highly red-fluorescent resorufin. [4] In 96-well microplates, 90 μL/well of the inoculum were mixed with 1.6 μL/well of each concentration of each compound and 8.4 μL/well of the liquid medium. Amphotericin B and rifampicin were used as positive and negative controls, respectively. The plates were incubated at 37 °C for 25-30 h without agitation. After incubation, fluorescence was recorded on a multimode plate reader (EnVision Perkin Elmer) using wavelength settings for resorufin (excitation 570 nm, emission 600 nm). Growth inhibition was calculated using the equation mentioned above but using the fluorescence instead of absorbance. Wells with 0.002% resazurin in broth medium were used as blanks.

Cell viability was assessed by CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (Promega) following the manufacturer's instructions. The assay contains MTS (3-(4,5- 14. C.elegans starvation recovery assay Starvation recovery was examined using the nematode Caenorhabditis elegans N2 Bristol strain obtained from the Caenorhabditis Genetics Centre (CGC). We kept eggs in 0.5ml aliquots of liquid media (S-buffer: 5.85 g NaCl, 1.123 g K2HPO4, 5.926 g KH2PO4 per 1 l dH2O) without any nutrients, hence starving and arresting development of the nematodes at the L1 larvae stage. The liquid media was supplemented with either 10 μM, 25 μM or 50 μM of euglenatide B diluted in DMSO to a final concentration of 1% DMSO in the S-buffer, and a DMSO control with no euglenatide was also included. The starvation vials where kept at 20 °C and inverted twice daily for aeration.
We ran three separate starvation experiments when worms were starved either for 10 days, 20 days or 30 days. We used three replicate tubes containing 100 eggs each per treatment (i.e. euglenatide concentration) for each starvation experiment. After starvation we transferred the worms onto standard Nematode Growth Medium (NGM) plates seeded with Escherichia coli OP50 as a food source. We counted the number of viable reproductive adults after four, five and six days on food. For statistical analyses, we used the total number of sexually mature adults recovered on plates by day six. The presence of euglenatide in the media reduced the number of viable adults with increasing concentration of euglenatide in 10-day starvation (Kruskal-Wallis test: EG:JM medium. Inocula preparation, cultivation and extraction were performed as described above. Extracts were analysed by a Shimadzu LC-PDA-MS as described above. The LC and MS data were processed using ACD/Spectrus, and the MS/MS spectra were converted to an mzXML file format using LabSolutions software (Shimadzu). GNPS spectral library [6] was searched for similar molecules using the converted files of MS/MS spectra (GNPS; https://gnps.ucsd.edu) and used to construct a molecular network of euglenatide-related metabolites. The following GNPS parameters were used: [7] Parent Mass Tolerance = 1 Da, Min Pairs Cos = 0.6, Min Matched Peaks = 3, Network TopK = 15, MSCluster = ON, Minimum Peak Intensity = 25, Filter Precursor Window = OFF, Filter Library = OFF, and Filter peaks in 50 Da Window = OFF. The molecular network was exported from GNPS and analysed using the visualisation software Cytoscape. [8] The GNPS job can be found on the following linkhttps://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=8c575ac25fb8422aaffd212ea e5c1b15. The MASSIVE dataset is available to the public with the following name and number: Euglenatides from Euglena species MSV000088616.

Figure S23
Comparison of CD spectra between euglenatide E and those reported for nemamide A and triene diastereomers. [9]

Figure S28
Comparative total absorbance (200-600 nm) and total ion chromatograms of E. mutabilis cultivated in EG:JM medium.