Rare Glutamic Acid Methyl Ester Peptaibols from Sepedonium ampullosporum Damon KSH 534 Exhibit Promising Antifungal and Anticancer Activity

Fungal species of genus Sepedonium are rich sources of diverse secondary metabolites (e.g., alkaloids, peptaibols), which exhibit variable biological activities. Herein, two new peptaibols, named ampullosporin F (1) and ampullosporin G (2), together with five known compounds, ampullosporin A (3), peptaibolin (4), chrysosporide (5), c(Trp-Ser) (6) and c(Trp-Ala) (7), have been isolated from the culture of Sepedonium ampullosporum Damon strain KSH534. The structures of 1 and 2 were elucidated based on ESI-HRMSn experiments and intense 1D and 2D NMR analyses. The sequence of ampullosporin F (1) was determined to be Ac-Trp1-Ala2-Aib3-Aib4-Leu5-Aib6-Gln7-Aib8-Aib9-Aib10-GluOMe11-Leu12-Aib13-Gln14-Leuol15, while ampullosporin G (2) differs from 1 by exchanging the position of Gln7 with GluOMe11. Furthermore, the total synthesis of 1 and 2 was carried out on solid-phase to confirm the absolute configuration of all chiral amino acids as L. In addition, ampullosporin F (1) and G (2) showed significant antifungal activity against B. cinerea and P. infestans, but were inactive against S. tritici. Cell viability assays using human prostate (PC-3) and colorectal (HT-29) cancer cells confirmed potent anticancer activities of 1 and 2. Furthermore, a molecular docking study was performed in silico as an attempt to explain the structure-activity correlation of the characteristic ampullosporins (1–3).


Isolation and Structural Elucidation of Compounds 1-7
The chromatographic separation of the culture broth and mycelial crude extract using Diaion HP 20 and Sephadex LH 20 in combination with (semi)preparative HPLC yielded seven compounds (1−7) (Figure 1).
Compound 1 was isolated as a white, amorphous solid. The amino acid sequence of 1 was determined based on positive and negative ion ESI-HRMS n studies, which showed diagnostic fragments of the b and y series (Figures 2 and 3; Table 1; Table S1, Supplementary Materials).          13 − and y 14 − representing Ala 2 and Ac-Trp 1 as the acetylated N-terminal amino acid. In addition, the observed series of C-terminal ions y 4 + to y 12 + and y 9 − to y 12 − fully supported the sequence deduced from b series. Thus, the N-terminal peptide part was shown to be Ac-Trp 1 -Ala 2 -Aib 3 -Aib 4 -Lxx 5 -Aib 6 -Gln 7 -Aib 8 -Aib 9 -Aib 10 -GluOMe 11 -Lxx 12 -Aib 13 .
The sequence of 1 resulting from mass spectrometry fragmentations was confirmed by 1D and 2D NMR data, which simultaneously specified the isomeric residues Leu and Ile.
As depicted in Figure 4, 1 H-1 H correlations from TOCSY and COSY spectra allowed defining eight N-H doublet peaks as part of eight spin systems, of which five correspond to one Ala, two Leu, and two Gln residues. Another leucine-related coupling system showing additional hydroxymethylene signals at δ H 3.30/3.18, (δ C 63.5) and a hydroxyl resonance at δ H 4.50 bound to its methine at δ H 3.78 (δ C 48.2), demonstrated the presence of the reduced C-terminal Leuol residue. An additional moiety was pointed out by 1 H-1 H TOCSY correlations between the N-H amide group at δ H 8.38, the methine at δ H 4.40 (δ C 54.4), and the methylene at δ H 3.12/2.98, (δ C 26.8). 1 H-13 C HMBC correlations demonstrated that this methylene carbon is linked to protons of the indole ring. Additionally, the ROESY correlation between the above amide at δ H 8.38 and the acetyl CH 3 group at δ H 1.86 (δ C 22.2), which showed HMBC correlation to a carbonyl carbon at δ C 170.2, clearly resulted in the characterization of an acetylated N-terminal Trp residue. Finally, the δ-methylene of a glutamine-related spin system exhibited HMBC correlation to a carbonyl carbon at δ C 173.0, which itself showed a strong HMBC correlation with a methoxy group at δ H 3.55 (δ C 50.8), supporting the presence of a glutamic acid δ-methyl ester residue. Detailed analysis of HMBC interactions of N-H resonances with carbonyl and C-α signals coupled with ROESY correlations between neighboring proton signals afforded the sequence establishment as in accordance with mass spectrometry fragmentations. Therefore, the structure of 1 was established as Ac-Trp 1 -Ala 2 -Aib 3 -Aib 4 -Leu 5 -Aib 6 -Gln 7 -Aib 8 -Aib 9 -Aib 10 -GluOMe 11 -Leu 12 -Aib 13 -Gln 14 -Leuol 15 , and named as ampullosporin F (1) consistent with the ampullosporin series reported by Ritzau et al. in 1997 [21] and Kronen et al. in 2001 [22]. Detailed analysis of HMBC interactions of N-H resonances with carbonyl and C-α signals coupled with ROESY correlations between neighboring proton signals afforded the sequence establishment as in accordance with mass spectrometry fragmentations. Therefore, the structure of 1 was established as Ac-Trp 1 -Ala 2 -Aib 3 -Aib 4 -Leu 5 -Aib 6 -Gln 7 -Aib 8 -Aib 9 -Aib 10 -GluOMe 11 -Leu 12 -Aib 13 -Gln 14 -Leuol 15 , and named as ampullosporin F (1) consistent with the ampullosporin series reported by Ritzau et al. in 1997 [21] and Kronen et al. in 2001 [22].
Compound 7 was obtained as a white, amorphous solid. On the basis of ESI-HRMS studies, the molecular formula C 14 7), which was first synthesized in 1998 [55] and later recognized in different fungal sources such as Eurotium sp. [56] and Eurotium chevalieri MUT 2316 [57]. To the best of our knowledge, this is the first detection of c(Trp-Ser) (6) and c(Trp-Ala) (7) in a Sepedonium species.

In Situ Chemical Analysis
Because ampullosporin F (1) and G (2) were isolated as minor compounds with similar structures as ampullosporin A (3), the dominant component of S. ampullosporum, a question may arise with regard to the authenticity of these new compounds 1 and 2. Are the isolated ampullosporins 1 and 2 biosynthesized by fungus itself or are they artefacts formed during extraction, fractionation and/or the purification processes using methanol?
In order to answer this question, a crude extract from cultivated S. ampullosporum was prepared using ethanol, instead of methanol, and investigated using LC-HRMS screening approach. From an enriched fraction of the ethanol crude extract of S. ampullosporum, the ampullosporin F (1) and G (2) as well as ampullosporin A (3) could be unambiguously detected by their characteristic mass ( Figure S43, Supplementary Materials). Therefore, ampullosporin F (1) and G (2) are native constituents of S. ampullosporum Damon strain KSH 534.

Solid Phase Synthesis and Absolute Configuration of Ampullosporin F (1) and G (2)
The absolute configuration of 1 and 2 was established based on solid-phase peptide synthesis using Fmoc protected, L-configured amino acids (except the achiral Aib) and tetramethylfluoroform-amidinium hexafluorophosphate (TFFH) as a coupling reagent (Scheme 1).
The combined manual and automated synthesis was carried out on L-Leucinol 2-chlorotrityl polystyrene resin. The first four amino acids residues were incorporated by automated synthesis using the standard PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate) strategy, while the rest of the synthesis was performed manually using TFFH or HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) activations. N-terminal acetylation was achieved after complete peptide assembly by treating the resin with Ac 2 O, followed by solid-phase cleavage and global deprotection using TFA. The synthesized peptaibols 1 and 2 were purified by size exclusion column chromatography using Sephadex LH20 in combination with preparative HPLC. The ESI-HRMS n , 1D NMR, and CD spectra of the synthetic peptaibols 1 and 2 were consistent with those of the natural ampullosporin F (1) and G (2) (Figures S17-S28, Supplementary Materials). Consequently, all chiral amino acids naturally present in 1 and 2 possess the L-configuration, which is typical for the particular class of ampullosporins [21,35].

Solid Phase Synthesis and Absolute Configuration of Ampullosporin F (1) and G (2)
The absolute configuration of 1 and 2 was established based on solid-phase peptide synthesis using Fmoc protected, L-configured amino acids (except the achiral Aib) and tetramethylfluoroform-amidinium hexafluorophosphate (TFFH) as a coupling reagent (Scheme 1). Scheme 1. Solid-phase peptide synthesis of ampullosporin F (1) and G (2).
The combined manual and automated synthesis was carried out on L-Leucinol 2chlorotrityl polystyrene resin. The first four amino acids residues were incorporated by automated synthesis using the standard PyBOP (benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate) strategy, while the rest of the synthesis was performed manually using TFFH or HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) activations. N-terminal acetylation was achieved after complete peptide assembly by treating the resin with Ac2O, followed by solid-phase cleavage and global deprotection using TFA. The synthesized peptaibols 1 and 2 were purified by size exclusion column chromatography using Sephadex LH20 in combination with preparative HPLC. The ESI-HRMS n , 1D NMR, and CD spectra of the synthetic peptaibols 1 and 2 were consistent with those of the natural ampullosporin F (1) and G (2) (Figures S17-S28, Supplementary Materials). Consequently, all chiral amino acids naturally present in 1 and 2 possess the L-configuration, which is typical for the particular class of ampullosporins [21,35].

Evaluation of Antifungal Activities
Isolated peptaibols 1-3 were examined for their antifungal activities against plant pathogenic ascomycetous fungi Botrytis cinerea (grey mold pathogen on many crops, e.g., strawberries and wine grapes) and Septoria tritici (causes septoria leaf blotch of wheat) as well as the oomycete Phytophthora infestans (causal agent of the late blight disease on potato and tomato) using a 96-well microtiter plate assay. The commercially available fungicides epoxiconazole and terbinafine were used as positive controls.
All three compounds 1-3 showed strong activity against B. cinerea and only slightly lower inhibitory effects against P. infestans, but were inactive against S. tritici. Interestingly, the growth-inhibitory effects against both B. cinerea and P. infestans increased with the presence of GluOMe in ampullosporin F (1) and G (2), compared to the ampullosporin A (3) (Table 4, Figure S41, Supplementary Materials).

Evaluation of Anticancer Activities
The peptaibols 1-3 were tested for their effects on the viability of two different human cancer cell lines, namely prostate PC-3 adenocarcinoma cells and colorectal HT-29 adenocarcinoma cells. The cell viability and cytotoxicity assay was conducted by using resazurin and fluorometric read-out after 48 h cell treatment. The saponin digitonin (100 µM), a very potent permeabilizer of cell membranes, was used as positive control compromising the cells to yield 0% cell viability after 48 h. As negative control, representing 100% cell viability for data normalization, medium with 0.5% (v/v) DMSO supplementation (highest final DMSO concentration in test item samples) was measured. The peptaibols 1-3 were tested with concentrations in the range of 0.195-100 µM (factor 2 dilutions) in order to determine IC 50 values that have been calculated to be~3-6 µM for both novel ampullosporins F (1) and G (2), with twofold higher activity in prostate PC-3 cancer cells compared to the colorectal cancer cells HT-29. Furthermore, in both cancer cell lines, 1 and 2 were found with twofold lower IC 50 s compared to the already known ampullosporin A (3) (see Figure 5), indicating that the GluOMe modifications at the amino acid positions 7 and 11, respectively, enhance not only the antifungal but also the anticancer activity of the ampullosporins. Summarized results are shown in Table 4.

In Silico Molecular Docking
In 2009, Berek et al. investigated the neuroleptic-like activity of ampullosporin A (3) in mice. Thereby, they described a complete suppression of the effects of the N-methyl-Daspartate (NMDA) receptor antagonist MK-801, and alteration of the activity of those glutamate receptors [23], indicating NMDA receptors as potential biological target molecules of ampullosporin A (3). Recently, Lu et al. (2017) investigated the interplay of MK-801 and NMDA receptor in more detail using cryo-EM structural analyses, explaining the allosteric antagonistic action of MK-801 (pdb-code 5UOW) [58]. Based on those results, we decided to proof the hypothesis of NMDA receptor binding of our ampullosporins (1-3) by using a chemoinformatic molecular docking approach based on two protein databank entries (5UOW and 6IRA). While 5UOW includes the MK-801 inhibitor but reflects the situation at nonhuman NMDA receptor proteins, 6IRA comprises the human GluN1/GluN2A ligand binding domain as the relevant part of the human NMDA receptor [59].
Based on the protein databank entry 5UOW, a putative binding site on the triheteromeric NMDA receptor GluN1/GluN2A/GluN2B was indicated by the cryo-EMbased localization of the inhibitor MK-801 in structure 5UOW ( Figure 6). Moreover, our in silico docking approach based on 5UOW highlighted a very good docking of 3 in very close proximity to MK-801, as also shown in Figure 6. The indicated binding site is located in a hydrophobic cleft that is formed by several helices of both NMDA receptor subunits GluN1 and GluN2A. This theoretical co-localization of ampullosporin A (3) and the allosteric NMDA receptor antagonist MK-801 could explain the complete suppression of MK-801 effects caused by the peptaibol (3).   [58]. Based on those results, we decided to proof the hypothesis of NMDA receptor binding of our ampullosporins (1-3) by using a chemoinformatic molecular docking approach based on two protein databank entries (5UOW and 6IRA). While 5UOW includes the MK-801 inhibitor but reflects the In addition, compounds 1-3 were tested for their antiproliferative effects in PC-3 prostate and HT-29 colorectal human cancer cells, which, to some extent, express the NMDA receptor proteins GluN1 and GluN2A (according to mRNA expression data; analyzed by using the Genevestigator software, data base HS_mRNASeq_HUMAN_GL-1; data not shown). Therefore, we also investigated the ampullosporins' binding in silico based on protein databank entry 6IRA representing the human GluN1/GluN2A NMDA receptor complex that, however, do not include MK-801. Indeed, also in our 6IRA-based in silico model the peptaibols 1-3 were docked with best scores at the same position in the hydrophobic cleft between GluN1 and GluN2A (Figure 7). calization of the inhibitor MK-801 in structure 5UOW ( Figure 6). Moreover, our in silico docking approach based on 5UOW highlighted a very good docking of 3 in very close proximity to MK-801, as also shown in Figure 6. The indicated binding site is located in a hydrophobic cleft that is formed by several helices of both NMDA receptor subunits GluN1 and GluN2A. This theoretical co-localization of ampullosporin A (3) and the allosteric NMDA receptor antagonist MK-801 could explain the complete suppression of MK-801 effects caused by the peptaibol (3). In addition, compounds 1-3 were tested for their antiproliferative effects in PC-3 prostate and HT-29 colorectal human cancer cells, which, to some extent, express the NMDA receptor proteins GluN1 and GluN2A (according to mRNA expression data; analyzed by using the Genevestigator software, data base HS_mRNASeq_HUMAN_GL-1; data not shown). Therefore, we also investigated the ampullosporins' binding in silico based on protein databank entry 6IRA representing the human GluN1/GluN2A NMDA receptor complex that, however, do not include MK-801. Indeed, also in our 6IRA-based in silico model the peptaibols 1-3 were docked with best scores at the same position in the hydrophobic cleft between GluN1 and GluN2A (Figure 7). Our in silico results, coupled with the observations by Berek et al. [23], led to the hypothesis that this hydrophobic cleft at the interphase between GluN1 and GluN2A could be the binding site for the very hydrophobic peptaibols 1-3, causing their biological effects. To shed more light on the molecular ampullosporins binding mode and especially to investigate consequences of the sequence modifications in the novel ampullosporin F (1) and G (2), further docking studies were performed to validate this hydrophobic cleft as a potential binding site of compounds 1-3.

In Silico Molecular Docking
As illustrated in Figure 8 and Figure S42 (Supplementary Materials), all compounds 1-3 fit nicely into the described hydrophobic cleft and seem to be stabilized by numerous hydrophobic interactions to hydrophobic receptor side chains. Accordingly, for ampullosporin A (3) an interaction energy of −92.0 kcal/mol was calculated. However, replacement of each of Gln residues (Gln 7 and Gln 11 ) in ampullosporin A (3) by GluOMe resulted in additional hydrophobic interactions of ampullosporin F (1; GluOMe 11 ) and ampullosporin G (2; GluOMe 7 ) with hydrophobic receptor residues. In case of 1, enhanced hydrophobic Our in silico results, coupled with the observations by Berek et al. [23], led to the hypothesis that this hydrophobic cleft at the interphase between GluN1 and GluN2A could be the binding site for the very hydrophobic peptaibols 1-3, causing their biological effects. To shed more light on the molecular ampullosporins binding mode and especially to investigate consequences of the sequence modifications in the novel ampullosporin F (1) and G (2), further docking studies were performed to validate this hydrophobic cleft as a potential binding site of compounds 1-3.
As illustrated in Figure 8 and Figure S42 (Supplementary Materials), all compounds 1-3 fit nicely into the described hydrophobic cleft and seem to be stabilized by numerous hy-drophobic interactions to hydrophobic receptor side chains. Accordingly, for ampullosporin A (3) an interaction energy of −92.0 kcal/mol was calculated. However, replacement of each of Gln residues (Gln 7 and Gln 11 ) in ampullosporin A (3) by GluOMe resulted in additional hydrophobic interactions of ampullosporin F (1; GluOMe 11 ) and ampullosporin G (2; GluOMe 7 ) with hydrophobic receptor residues. In case of 1, enhanced hydrophobic interactions to the receptor residues V820, I824 and F637 elevate the calculated interaction energy to be −94.5 kcal/mol. In case of 2, the molecular docking indicates additional hydrophobic interactions of its GluOMe 7 modification with the hydrophobic receptor residues W608 and W611. The calculated interaction energy (−92.9 kcal/mol) for 2 is slightly enhanced compared to 3. Based on these calculated interaction energies, the binding of both ampullosporin F (1) and G (2) should outperform the binding of ampullosporin A (3). could be the binding site for the very hydrophobic peptaibols 1-3, causing their biological effects. To shed more light on the molecular ampullosporins binding mode and especially to investigate consequences of the sequence modifications in the novel ampullosporin F (1) and G (2), further docking studies were performed to validate this hydrophobic cleft as a potential binding site of compounds 1-3.
As illustrated in Figure 8 and Figure S42 (Supplementary Materials), all compounds 1-3 fit nicely into the described hydrophobic cleft and seem to be stabilized by numerous hydrophobic interactions to hydrophobic receptor side chains. Accordingly, for ampullosporin A (3) an interaction energy of −92.0 kcal/mol was calculated. However, replacement of each of Gln residues (Gln 7 and Gln 11 ) in ampullosporin A (3) by GluOMe resulted in additional hydrophobic interactions of ampullosporin F (1; GluOMe 11 ) and ampullosporin G (2; GluOMe 7 ) with hydrophobic receptor residues. In case of 1, enhanced hydrophobic interactions to the receptor residues V820, I824 and F637 elevate the calculated interaction energy to be −94.5 kcal/mol. In case of 2, the molecular docking indicates additional hydrophobic interactions of its GluOMe 7 modification with the hydrophobic receptor residues W608 and W611. The calculated interaction energy (−92.9 kcal/mol) for 2 is slightly enhanced compared to 3. Based on these calculated interaction energies, the binding of both ampullosporin F (1) and G (2) should outperform the binding of ampullosporin A (3).  Interestingly, the determined antiproliferative activities of compounds 1-3 ( Figure 5) correlate very well with the proposed enhanced binding of both novel ampullosporins (1 and 2). The antiproliferative IC 50 values in the anticancer assay were detected to be by twofold better for ampullosporin F (1) and G (2) than for ampullosporin A (3).
Whether or not the NMDA receptor is really the relevant molecular target explaining the observed antiproliferative effects of the ampullosporins on human cancer cells, further investigations are necessary. However, considering the well-published neuroleptic-like activities of ampullosporin A (3) [21,23,60] that can be easily attributed to NMDA receptor pathways, it would be of high interest to test our novel ampullosporins F (1) and G (2), with presumed improved NMDA receptor binding for their neuroleptic activity, e.g., in vivo in mice.
The high-resolution mass spectra in positive and negative modes were obtained from an Orbitrap Elite mass spectrometer (Thermofisher Scientific, Bremen, Germany) equipped with an ESI electrospray ion source (spray voltage 4.0 kV; capillary temperature 275 • C, source heater temperature 40 • C; FTMS resolution 60.000). Nitrogen was used as sheath gas. The sample solutions were introduced continuously via a 500 µL Hamilton syringe pump with a flow rate of 5 µL/min. The instrument was externally calibrated by the Pierce ® LTQ Velos ESI positive ion calibration solution (product number 88323) and Pierce ® ESI negative ion calibration solution (product number 88324) from Thermofisher Scientific (Rockford, IL, USA). The data were evaluated by the Xcalibur software 2.7 SP1 Thermofisher Scientific, Waltham, MA, USA). The collision induced dissociation (CID) MS n measurements were performed using the relative collision energies given in Table S1 (Supplementary Materials).

Fungal Strain and Cultivation
The fungal strain Sepedonium ampullosporum Damon KSH 534 was isolated in August 1999, from Boletus calopus in Crista Acri near Cosenza, Italy (leg./det. C. Lavorato). A voucher specimen is deposited at the herbarium of the University Regensburg. The fungal culture of S. ampullosporum strain KSH 534 was stored on malt peptone agar (MPA) plates and transferred periodically. The upscaled semi-solid cultures, used for isolation, were grown in 31 Erlenmeyer flasks (size 1 L) each containing 1.5 g of cotton wool and 250 mL of malt peptone medium (2.5 g malt and 0.625 g peptone in 250 mL deionized water), resulting in a total volume of 7.75 L. Each culture flask was inoculated with a 10 × 10 mm agar plug of colonized fungus and incubated for 14 days at room temperature without agitation.

Sample Preparation for LC-MS Screening
For the preparation of the enriched fraction for the LC-HRMS screening, one stored deep frozen agar plate cultures of S. ampullosporum Damon strain KSH534 was crushed in small pieces and extracted with EtOH 96% (2 × 250 mL) in an ultrasonic bath at room temperature. The resulted yellow solution was evaporated in vacuo to dryness. The dried crude extract was redissolved in EtOH 96%/H 2 O (1:2, v/v) to a concentration of 50 mg/mL. The resulting solution was separated on SPE cartridges Chromabond ® C18 (loading 200 mg/3 mL, particle size 45 µm; Macherey-Nagel, Düren, Germany), targeted peptaibols 1-3 were eluted with EtOH 96%. After evaporation to dryness in vacuo, the enriched fraction was redissolved in CH 3 CN and submitted to LC-HRMS.

Solid-Phase Peptide Synthesis
Compounds 1 and 2 were synthesized by combining manual and automated synthesis. The protocol was based on a Fmoc/t-butyl strategy in a 0.1 mmol scale starting from L-Leucinol 2-chlorotrityl resin (200-400 mesh, loading 0.67 mmol/g resin). The first four amino acid residues were incorporated by automated synthesis using the standard method based in PyBOP/NMM activation. The rest of the synthesis was performed manually using 4 equiv. of the amino acids and DMF as solvent. For all the Aib residues, the coupling cycle protocol was based on activation with TFFH (4 equiv.) and NMM (N-methylmorpholine, 4 equiv.) for 12 min and coupling time of 120 min. The rest of the amino acids were coupled using activation with HATU (4 equiv.) and NMM (4 equiv.) for 5 min and a coupling time of 120 min. Fmoc removals were carried out using a solution of 20% piperidine in DMF for two cycles of 10 min. N-terminal acetylation was performed after complete peptide assembly by treating the resin with Ac 2 O (10 equiv) and DIPEA (10 equiv) in DMF for 30 min. Solid-phase cleavage and global deprotection was achieved by treating the resin with 5 mL of TFA/H 2 O/TIPS (95:2.5:2.5, v/v/v) for 120 min. The cleavage mixture was concentrated under reduced pressure, suspended in a mixture of CH 3 CN 50% in water and lyophilized.
Natural ampullosporin F ( Antiproliferative and cytotoxic effects, respectively, of the compounds 1-3 were investigated by performing a fluorimetric resazurin-based cell viability assay (Sigma-Aldrich, Taufkirchen, Germany). For that purpose, prostate PC-3 and colorectal HT-29 cancer cells were seeded in low densities into 96-well plates (3000-6000 cells per well; seeding conflu-ency~10%) and were allowed to adhere for 24 h. Subsequently, the cells were treated for 48 h with compound concentrations up to 100 µM. For control measurements, cells were treated in parallel with 0.5% DMSO (negative control, representing the final DMSO content of the highest concentrated test compound concentration) and 100 µM digitonin (positive control, for data normalization set to 0% cell viability), both in standard growth medium. As soon as the 48-h incubation was finished, the incubation medium was discarded, and cell were rinsed once with PBS. Resazurin solution in RPMI 1640 without phenol red and other supplements was prepared freshly prior to use, and added to the cells in a final resazurin concentration of 50 µM. Subsequently, the cells were incubated under standard growth conditions for further 2 h. Finally, the conversion of resazurin to resorufin by viable, metabolically active cells was measured with 540 nm excitation and 590 nm emission settings by using a SpectraMax M5 multiwell plate reader (Molecular Devices, San Jose, CA, USA). Data were determined in biological quatruplicates, each with technical triplicates. For data analyses GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2013 (Microsoft, Redmond, WA, USA) were used.

Computational Details
The X-ray structures of the human GluN1/GluN2A NMDA receptor in the glutamate/ glycine-bound state at pH 7.8 (pdb-code 6IRA) [59] and nonhuman (frog and mouse) triheteromeric NMDA receptor GluN1/GluN2A/GluN2B in complex with glycine, glutamate, the uncompetitive NMDA receptor antagonist MK-801 and a GluN2B-specific Fab, at pH 6.5 (pdb-code 5UOW) [58] were downloaded from the protein databank [61] and used for in silico docking analyses of compounds 1-3.
All theoretical investigations were performed using the molecular modeling software package MOE (molecular operating environment) [62]. The X-ray structure was prepared for docking studies by adding the missing hydrogen atoms using the 3D-protonate module implemented in MOE.
The putative active site of the enzyme is indicated by the co-crystallized inhibitor MK-801 (see Figure 6, left site). By closer inspection of this site, an almost complete hydrophobic cleft formed by several helices could be detected, which led to the hypothesis that this could be a perfect binding site for the very hydrophobic peptaibols due to multiple Aib amino acid containing residues. Therefore, docking studies were performed defining this as a binding site. For each of the three peptaibols, ampullosporin A (3), F (1), and G (2), 30 poses were generated using the triangle matcher for fast placement, the London dG as fitness function with subsequent induced fit relaxation of the binding site. The best scored docking poses are displayed and discussed.

Conclusions
In conclusion, the present study represents the chemical investigation of semi-solid culture of Sepedonium ampullosporum Damon strain KSH 534. There are seven constituents including two new 15-residue linear peptaibols, named ampullosporin F (1) and G (2), as well as two known linear peptaibols, ampullosporin A (3) and peptaibolin (4), together with three previously described cyclic peptides, chrysosporide (5), c(Trp-Ser) (6), and c(Trp-Ala) (7). The authenticity of peptaibols 1 and 2 was approved by using LC-HRMS approach. Additionally, the total synthesis of 1 and 2 was performed on solid-phase synthesis establishing the L-configuration of all chiral amino acids. Furthermore, peptaibols 1-3 showed significant anti-phytopathogenic activity against B. cinerea and P. infestans, but no activity against S. tritici. Moreover, compounds 1-3 exhibited strong anticancer activities against human prostate (PC-3) and colorectal (HT-29) cancer cells. Interestingly, for both antifungal and anticancer assays, the activities of ampullosporin F (1) and G (2) were found to be twofold higher than the structurally similar ampullosporin A (3), demonstrating the effect of a GluOMe moiety on biological activity. Our molecular docking data on NMDA receptors suggests the better hydrophobic interaction of 1 and 2 than 3 with the hydrophobic cleft of the receptor, which might lead to the higher inhibitory effects of the new compounds 1 and 2 compared to 3.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.