Indole Diketopiperazine Alkaloids from the Marine Sediment-Derived Fungus Aspergillus chevalieri against Pancreatic Ductal Adenocarcinoma

A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC50 values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.


Introduction
Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic neoplasm, is considered the fourth leading cause of cancer-related mortality worldwide due to its high aggressiveness and malignancy with a poor survival rate [1,2].Despite breakthroughs in diagnostic tools, surgical treatments, and chemotherapy, the overall survival rates of PDAC remain low, and the only available therapies are classical chemotherapeutics with an unselective cytotoxic profile.Consequently, there is an immense need to find new drugs or drug leads for curing this disease [2,3].Many natural products have been reported to exert anticancer effects against pancreatic cancer [3][4][5].As part of our ongoing search for discovering novel potential drug candidates for use against PDAC that originate from a natural source [6], a bioassay-guided isolation of secondary metabolites was conducted on a marine sediment-derived fungus, Aspergillus chevalieri, collected from the Red Sea at a depth of 15 m close to the lighthouse of Dahab, Egypt.Accordingly, seven prenylated indole diketopiperazine alkaloids, including a rare tetraprenylated derivative that has been reported only once before, were isolated from the bio-active fraction, identified, and further pharmacologically characterized [7,8].Indole diketopiperazine alkaloids (DKPs) represent a group of secondary metabolites broadly distributed in filamentous fungi, especially in the genera Aspergillus and Penicillium [9].Biosynthetically, in fungi, the core cyclic dipeptide nucleus of DKPs is mostly originated from the condensation of two tryptophan amino acids or one tryptophan molecule and another amino acid, assembled by non-ribosomal peptide synthetases (NRPSs) [8,10].Different putative tailoring enzymes contribute to the structural diversity of DKP scaffolds, including oxidoreductases, hydrolases, methyl transferases, prenyltransferases, and ligases [11,12].Prenylated indole diketopiperazines are constituted of an indole diketopiperazine backbone and isoprenoid moieties, in which prenylation is catalyzed by prenyltransferases acting as tailoring enzymes [7,8,13].The subsequent prenylation cascade of echinulin and neoechinulin series of alkaloids in Aspergillus ruber has been demonstrated to be mediated via two prenyltransferases, EchPT1 and EchPT2.These two enzymes are involved in the prenylation and reverse prenylation cascades taking place at different positions of the indole DKP core structure of echinulin and neoechinulin congeners, resulting in the formation of mono-, di-, tri-, and tetraprenylated derivatives [7,8].Indole DKPs are not only characterized by the diversities in their chemical structures but are also reported to exhibit significant biological activities, such as antiinflammatory, anticancer, antiviral, anti-neurogenerative, and antioxidant activities [9,14].Various indole DKPs have been evaluated for their anticancer activities against several cancer lines such as HL-60, P388, BEL-7402, A-549, PC12, and HeLa cells via induction of apoptosis and/inhibition of cell proliferation [14][15][16][17][18]. Owing to their improved lipophilicity, prenylated natural products are proven to possess better binding affinity to target proteins and better bioavailability compared to nonprenylated derivatives, creating a direct impact on the biological activity [19,20].Therefore, prenylated DKPs have demonstrated to be possible candidates for drug development and discovery [21,22].In the current study, we report the isolation of different prenylated indole diketopiperazine alkaloids, including a new triprenylated derivative rubrumline P (1) and a rare tetraprenylated known congener 1Q2 (7), from a marine sediment-derived fungus, A. chevalieri, through a bioassay-guided fractionation.In addition, the crystal structure of a known derivative, rubrumline I (2), is reported for the first time.Genome sequencing of the fungus revealed secondary metabolite regions presumably involved in the synthesis of the reported prenylated metabolites.Furthermore, an analysis of the isolated compounds' cytotoxic efficacy against pancreatic cancer cell line PANC-1 cells has been performed, followed by cell cycle analyses of the isolated compounds.

Bioactivity-Guided Fractionation of the Ethyl Acetate Extract of Aspergillus chevalieri
After cultivating A. chevalieri on a solid rice medium supplemented with 3.5% NaCl (mimicking the salinity of seawater), the crude ethyl acetate extract of the fungus was initially subjected to chromatographic separation via vacuum liquid chromatography.Subsequently, the produced VLC fractions were preliminary tested for their cytotoxic potential against the human pancreatic PANC-1 cancer cell line at a concentration of 100 µg/mL.Remarkable cytotoxic activity was observed for fractions 4 and 5 after 72 h compared to 1 µM of the microtubule inhibitor paclitaxel, part of the first-line treatment regimen for PDAC patients (Figure 1).Incubation of fraction 4 for 72 h reduced the viability of PANC-1 cells by about 99%.A comparable result was observed for fraction 5. Based on the bioassay result and HPLC profile of fraction 4 (100% EtOAc) compared to those Mar.Drugs 2024, 22, 5 3 of 14 of fraction 5 (90% DCM-MeOH), which was an oily fraction, fraction 4 was selected for further chromatographic separations to isolate and identify the active compounds.µg/mL.Remarkable cytotoxic activity was observed for fractions 4 and 5 after 72 h compared to 1 µM of the microtubule inhibitor paclitaxel, part of the first-line treatment regimen for PDAC patients (Figure 1).Incubation of fraction 4 for 72 h reduced the viability of PANC-1 cells by about 99%.A comparable result was observed for fraction 5. Based on the bioassay result and HPLC profile of fraction 4 (100% EtOAc) compared to those of fraction 5 (90% DCM-MeOH), which was an oily fraction, fraction 4 was selected for further chromatographic separations to isolate and identify the active compounds.

Structure Elucidation
After our chromatographic fractionation of the crude extract of A. chevalieri on VLC, various separations were conducted on other stationary phases, followed by final purifications utilizing semi-preparative HPLC affording seven indole diketopiperazine alkaloids, compounds 1-7 (Figure 2), including one new alkaloid (1) in addition to six known

Genome Sequencing
The genome assembly of Aspergillus chevalieri resulted in a total length of 29,419,542 bp, with an N50 fragment length of 1,488,407 bp and an average coverage of 3960×.The assembly consists of 192 contigs, of which 8 contigs contain more than 1,000,000 base pairs (Table 2).These eight contigs correspond to the eight chromosomes of the fungal genome as represented in the reference strain in the NCBI database (NCBI RefSeq assembly: GCF_016861735.1) [30].Regions coding for potential secondary metabolites were found on six of these eight contigs (Figure 5).Most were gene clusters for non-ribosomal peptide synthetases The specific optical rotation of compound 1 is negative, which is consistent with the negative values reported for other known neoechinulin A series of indole diketopiperazine alkaloids with 12S and 22S configurations, proposing that the new compound 1 shares the same absolute configuration as the known derivatives [23,24,29].Although the absolute configuration of the previously reported rubrumline derivatives was assigned using a modified Mosher's method and CD measurement, no crystal structure has been reported so far for these derivatives [23].Herein, we present an independent assignment of the absolute configuration of the known rubrumline I (2) via single-crystal X-ray diffraction analysis through anomalous dispersion for the first time, allowing an unambiguous assignment of the absolute configuration of 2 as 12S and 22S (Figure 4).Based on NMR data, optical rotations and biogenetic considerations, the same absolute configurations, as elucidated for compound 2 via X-ray diffraction analysis, can be assumed for the other alkaloids as well.

Genome Sequencing
The genome assembly of Aspergillus chevalieri resulted in a total length of 29,419,542 bp, with an N50 fragment length of 1,488,407 bp and an average coverage of 3960×.The assembly consists of 192 contigs, of which 8 contigs contain more than 1,000,000 base pairs (Table 2).These eight contigs correspond to the eight chromosomes of the fungal genome as represented in the reference strain in the NCBI database (NCBI RefSeq assembly: GCF_016861735.1) [30].
Regions coding for potential secondary metabolites were found on six of these eight contigs (Figure 5).Most were gene clusters for non-ribosomal peptide synthetases (NRPSs), which were found on five contigs, followed by regions for type I polyketide synthases (T1PKSs), terpenes, and fungal ribosomally synthesized and post-translationally modified peptide (RiPP)-like secondary metabolites, on two contigs each.Regions for NIsiderophones were also found on one contig.In an additional analysis with InterProScan, a potential region for terpenoid cyclases and protein prenyltransferases was also found on a 21,917 bp-long contig, which does not belong to the core genome.Analysis of the gene clusters in the genome of A. chevalieri revealed the presence of gene regions for the production of non-ribosomal peptide synthetases, polyketide synthases, and terpenes.There is also an evidence that this fungus is able to synthesize prenyltransferases.Both findings indicate that A. chevalieri has the ability to synthesize isoprenylated natural products.The synthesis of isoprenylated natural products through the formation of echinulin prenyltransferase 1 (EchPT1) has so far only been described for other members of the genus Aspergillus (A. fumigatus, A. oryzae, and A. ruber), but not yet for Aspergillus chevalieri [8,31,32].

Cytotoxicity and Cell Cycle Analyses in the Pancreatic Ductal Adenocarcinoma Cell Line PANC-1
Based on the remarkable cytotoxicity results from the bioactivity-guided screening of the starting fraction 4, the purified compounds 1 and 4 were characterized in full doseresponse curves in the selected pancreatic cancer cell line.The respective cytotoxic efficacy levels are displayed in Table 3.
Table 3. Cytotoxic efficacy in PANC-1 cells represented by the respective IC 50 values after 72 h of incubation.The arithmetic means of three independent biological repeats and their respective standard deviations are depicted.

Compound IC 50 (±SD) [µM]
PANC-1 In contrast to the standard-of-care treatment paclitaxel, for which IC 50 values in the low nanomolar range are reported [33], compounds 1 and 4 display only moderate cytotoxic efficacy levels, with IC 50 values of 25.8 and 23.4 µM, respectively.
To determine whether compound 1 or compound 4 affects the cell cycle of PANC-1 cells, a cell cycle analysis was performed.The tumor cells were treated with 30 µM of compound 1 or compound 4 for 24 h, respectively, and stained according to the Nicoletti method.Treatment with the spindle toxin paclitaxel served as a positive control.Treatment with compound 1 or compound 4 did not result in any significant changes in the cell cycle compared to the solvent control DMSO (Figure 6).However, a slight increase in the sub-G1 peak was observed after treatment with compounds 1 and 4, but this did not reach levels of statistical significance.In contrast, and as expected, treatment with 0.1 µM paclitaxel resulted in the arrest of the cell cycle in the G2 phase and a significant increase in the sub-G1 peak (Figure 6).Therefore, the cytotoxic effects of these compounds might be attributed to inducing apoptosis; however, the exact mechanism-of-action is still elusive.
(NRPSs), which were found on five contigs, followed by regions for type I polyketide synthases (T1PKSs), terpenes, and fungal ribosomally synthesized and post-translationally modified peptide (RiPP)-like secondary metabolites, on two contigs each.Regions for NIsiderophones were also found on one contig.In an additional analysis with InterProScan, a potential region for terpenoid cyclases and protein prenyltransferases was also found on a 21,917 bp-long contig, which does not belong to the core genome.For each of the six genome assembly contigs on which regions for secondary metabolites were identified, the position of the region and the structure type of the metabolites encoded there are shown.In addition, the most similar metabolite cluster already known is indicated with the associated cluster type and the degree of similarity (%).

Fungal Material
The fungus Aspergillus chevalieri LH15-100R2 was isolated from a marine sediment, which was collected from the Red Sea in November 2017 at a depth of 15 m near to the lighthouse of Dahab, Egypt.The fungus was identified as A. chevalieri (GenBank accession no.OR605556) through amplification and sequencing of the internal transcribed spacer region (ITS), including the 5.8S ribosomal DNA, followed by a BLAST search in NCBI, as described before [34].This fungal strain is stored at −80 • C in the Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, Düsseldorf.

Fermentation, Extraction, and Isolation
Fermentation of the fungus was carried out in ten 1L Erlenmeyer flasks, each containing 100 g of rice (Oryza Milchreis), 3.5 g of NaCl, and 110 mL of demineralized water.After autoclaving at 121 • C for 20 min then cooling to room temperature, the fungus was transferred to the solid rice medium.The fungal culture was left to grow under static conditions for 23 days at room temperature until the fungus had totally overgrown the medium.Afterwards, the solid fermented fungus was extracted two times with EtOAc (each with 600 mL).After soaking overnight in EtOAc, the solid medium was cut into small pieces and then shaken for 8 h at 150 rpm.The combined extracts were concentrated via evaporation of EtOAc under reduced pressure, yielding around 15 g of EtOAc crude extract.The crude extract (15 g) was fractionated through VLC (vacuum liquid chromatography) on silica gel as a stationary-phase with a gradient elution of solvents consisting of mixtures of n-hexane/EtOAc and CH 2 Cl 2 /MeOH, to afford seven fractions (A.ch Fr. 1-7).Fraction A. ch.Fr. 4 (640 mg), eluted with 100% EtOAc, was further purified via Sephadex LH20 column chromatography using CH 2 Cl 2 -MeOH (1:1) as a mobile phase, yielding three subfractions (A.ch Fr.

DNA Isolation and Genome Sequencing
DNA isolation was performed according to the Quick DNA fungal/bacterial miniprep protocol provided by Zymo Research [39].
For long-read Nanopore sequencing, the NBE_9169_v114_revH_15Sep2022 protocol for ligation sequencing of genomic DNA was followed, using the SQK-NBD114 native barcoding kit and a PromethION Flow Cell R10 Version.Reads were assembled using Flye Version 2.9.2-b1794 [40] with asmCoverage-Mode.Antismash [41] and InterProScan Version 5.58-91.0[42] were used to detect gene clusters and identify potential genes of interest.

Cell Cycle Analysis
For analysis of the cell cycles, 9 × 10 4 cells/mL of PANC-1 were cultured in 6-well plates (Greiner Bio-One).After treatment for 24 h and harvesting via trypsin, the cells were washed with D-PBS.For cell cycle analyses, the cells were suspended in 100 µL of hypotonic buffer (1% sodium citrate, 0.1% Triton X-100, and 50 µg/mL propidium iodide in double-distilled water).The suspension was incubated for 10 min at room temperature.Flow cytometry was performed with the BD FACSLyric flow cytometer (#87135, BD Biosciences, Heidelberg, Germany), whereby FlowJo software (version 10.8.1) was utilized for the analysis.

Figure 1 .
Figure 1.Results of the bioassay-guided fractionation: Cytotoxic effects of the vacuum liquid chromatography (VLC) fractions of Aspergillus chevalieri on PANC-1 cells.Cells were seeded into a 96well plate at a concentration of 5 × 10 3 cells/well and treated for 72 h after seeding with the different VLC fractions at a concentration of 100 µg/mL.A solvent control (1% DMSO) and a positive control (1 µM paclitaxel) were incubated for 72 h.Cell viability is expressed as the percentage of untreated cells.Error bars indicate standard errors of the mean (SEM) (n = 1 with three technical replicates per subjected fraction).

Figure 1 .
Figure 1.Results of the bioassay-guided fractionation: Cytotoxic effects of the vacuum liquid chromatography (VLC) fractions of Aspergillus chevalieri on PANC-1 cells.Cells were seeded into a 96-well plate at a concentration of 5 × 10 3 cells/well and treated for 72 h after seeding with the different VLC fractions at a concentration of 100 µg/mL.A solvent control (1% DMSO) and a positive control (1 µM paclitaxel) were incubated for 72 h.Cell viability is expressed as the percentage of untreated cells.Error bars indicate standard errors of the mean (SEM) (n = 1 with three technical replicates per subjected fraction).

Figure 2 .
Figure 2. Structures of the isolated compounds from Aspergillus chevalieri.

Figure 2 .
Figure 2. Structures of the isolated compounds from Aspergillus chevalieri.

Figure 4 .
Figure 4. Molecular structure of 2 from a single-crystal X-ray structure determination analysis (50% thermal ellipsoids, H atoms with arbitrary radii).See Figure S9 in the Supplementary Materials for the packing diagram and a diagram of the hydrogen-bonding network.

Figure 4 .
Figure 4. Molecular structure of 2 from a single-crystal X-ray structure determination analysis (50% thermal ellipsoids, H atoms with arbitrary radii).See Figure S9 in the Supplementary Materials for the packing diagram and a diagram of the hydrogen-bonding network.

Figure 5 .
Figure 5. Identified secondary metabolites regions for Aspergillus chevalieri.For each of the six genome assembly contigs on which regions for secondary metabolites were identified, the position of the region and the structure type of the metabolites encoded there are shown.In addition, the most similar metabolite cluster already known is indicated with the associated cluster type and the degree of similarity (%).

Figure 5 .
Figure 5. Identified secondary metabolites regions for Aspergillus chevalieri.For each of the six genome assembly contigs on which regions for secondary metabolites were identified, the position of the region and the structure type of the metabolites encoded there are shown.In addition, the most similar metabolite cluster already known is indicated with the associated cluster type and the degree of similarity (%).

Figure 6 .
Figure 6.Detection of cell cycle phases in PANC-1 cells.The cells were treated with 30 pound 1, 30 µM of compound 4, or 0.1 µM paclitaxel for 24 h.Cell cycle analysis wa according to the Nicoletti method.(a) Percentage of PANC-1 cells in the different cell c h of treatment with compound 1, compound 4, or paclitaxel, shown in explementary (b) Quantification of cell cycle phases.The cell cycles of at least 10,000 cells were analy cytometry.Error bars indicate the standard errors of the mean (n = three independent with * = p ≤ 0.05, and **** = p ≤ 0.0001).

Figure 6 .
Figure 6.Detection of cell cycle phases in PANC-1 cells.The cells were treated with 30 µM of compound 1, 30 µM of compound 4, or 0.1 µM paclitaxel for 24 h.Cell cycle analysis was performed according to the Nicoletti method.(a) Percentage of PANC-1 cells in the different cell cycles after 24 h of treatment with compound 1, compound 4, or paclitaxel, shown in explementary histograms.(b) Quantification of cell cycle phases.The cell cycles of at least 10,000 cells were analyzed via flow cytometry.Error bars indicate the standard errors of the mean (n = three independent experiments, with * = p ≤ 0.05, and **** = p ≤ 0.0001).

Table 2 .
Length and coverage of the Aspergillus chevalieri genome contigs and the identified secondary metabolite regions.

Table 2 .
Length and coverage of the Aspergillus chevalieri genome contigs and the identified secondary metabolite regions.