Cyclic Peptides from the Soft Coral-Derived Fungus Aspergillus sclerotiorum SCSIO 41031

Three novel cyclic hexapeptides, sclerotides C–E (1–3), and a new lipodepsipeptide, scopularide I (4), together with a known cyclic hexapeptide sclerotide A (5), were isolated from fermented rice cultures of a soft coral-derived fungus: Aspergillus sclerotiorum SCSIO 41031. The structures of the new peptides were determined by 1D and 2D NMR spectroscopic analysis, Marfey’s method, ESIMS/MS analysis, and single crystal X-ray diffraction analysis. Scopularide I (4) exhibited acetylcholinesterase inhibitory activity with an IC50 value of 15.6 μM, and weak cytotoxicity against the human nasopharyngeal carcinoma cell line HONE-EBV with IC50 value of 10.1 μM.


Introduction
Marine microorganisms are generally considered to be a significant new chemical resource of secondary metabolites [1,2]. These organisms thrive in the hostile and competitive oceanic environment and produce a variety of chemically diverse and biologically active compounds [3][4][5], which have attracted great attention in biomedical research [6,7]. As a class of important metabolites from marine microorganisms, cyclic peptides generally possess a scarce molecular skeleton. Representatives include lucentamycins [8] and marthiapeptide A [9] from marine actinomycetes, sclerotiotides A-K [10], JBIR-15 [11], maribasins [12] and sclerotides A and B [13] from marine fungi. Of these representatives, sclerotides present a unique hexapeptide containing both anthranilic acid and dehydroamino acid residues, which are rarely reported in nature.
In our ongoing studies discovering structurally novel and bioactive natural hybrid peptides from soft coral-derived fungi [14], three novel cyclic hexapeptides, sclerotides C-E (1-3) and a new lipodepsipeptide scopularide I (4), along with a known cyclic hexapeptide, sclerotide A (5) [13], were obtained from Aspergillus sclerotiorum SCSIO 41031 ( Figure 1). Herein we report the isolation, structure elucidation, and biological activities of these new cyclic peptides.
The connectivity between the residues of 3 was also established by the key HMBC correlations illustrated in Figure 2 9), revealing that the structure of 4 was similar to that of scopularide D [15]. Careful comparisons of the 1D and 2D NMR data between 4 and scopularide D showed that overall they were similar, but there was a slight difference which was the additional presence of one high-field methylene and the absence of a methyl attached at the long-chain fatty acid, which was in agreement with the 14 mass units difference. and the HMBC correlation (from H-25 to C-27). The complete structure for 4, including absolute configurations, was confirmed by single crystal X-ray analysis (CCDC 1816072) using Cu Kα radiation with Flack parameter of 0.05 (12), which allowed the assignments of amino acid residues as L-Val, L-Ala, D-Leu, and L-Val, and gave the configurations for (24S,25S)-24-hydroxyl-25-methyllauric acid (HMLA) lipid residue ( Figure 5).

Molecular Docking
In order to gain an insight into the molecular interactions between compounds 1-5 and AChE, the crystal structure of the torpedo californica acetylcholinesterase enzyme (PDB ID: 2CMF) [16] was used as the receptor, and it was subjected to an in silico molecular docking analysis with 1-5, using the induced-fit module in the Schrödinger software suite. As a result, compound 4 fit comfortably into the binding pocket for alkylene-linked tacrine dimers with similar binding positions. In the 2D binding model ( Figure 6B), the alkyl chain of 4 formed hydrophobic interaction with the active-site residues TRP84, ASP72, TRY70, and TRP279, and the NH of Gly formed a hydrogen bond with the active site residue TYR334. Compounds 1-3 and 5 were not beneficial for binding to AChE.

Mar. Drugs 2021, 19, x FOR PEER REVIEW
The connectivity between the residues of 3 was also established by the key correlations illustrated in Figure 2   Compound 4 was isolated as a colorless crystal, and HRESIMS data (m/z 652.4 + H] + ) supported a molecular formula of C34H61N5O7, accounting for 7 degrees of ration. The 1 H and 13 C NMR data ( Table 2) showed typical peptide characteristic

Fungal Material
The fungal strain SCSIO 41031 was isolated from a soft coral, which was collected in Beihai, Guangxi Province, China. The isolate was stored on Müller Hinton broth (MB) agar (malt extract 15.0 g, artificial sea salt 10.0 g, and agar 15.0 g) slants at 4.0 • C, and a voucher specimen was deposited in the CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China. Based on sequencing of the ITS region, the fungal strain SCSIO 41031 was identified as Aspergillus sclerotiorum with 100% similarity (GenBank no. KC478520.1).

Fermentation and Extraction
The strain Aspergillus sclerotiorum SCSIO 41031 was cultured on MB-agar plates at SCSIO 41031 was incubated for 30 days at room temperature in 1 L × 30 conical flasks with solid rice medium (each flask contained 200.0 g rice, 2.5 g artificial sea salt, and 250 mL H 2 O). The whole fermented cultures were overlaid and extracted with EtOAc three times to afford a brown extract (109 g).

Bioactivity Assay
The AChE inhibition activity was measured based on the modified Ellman's method [17]. Tacrine was used as positive drug. The inhibition rates of AChE were calculated using Origin 8.0 software.
The obtained compounds (4 and 5) were evaluated for their cytotoxic activities against three cancer cell lines, THP-1, HONE1, and HONE1-EBV. The THP-1, HONE1, and HONE1-EBV cell lines were obtained from Sun Yat-sen University Cancer Center. The cytotoxic activity was determined by the CCK-8 (Dojindo) method [19]. Briefly, THP-1, HONE1, and HONE1-EBV cells were cultured in DMEM media supplemented with 10% phosphatebuffered saline (FBS), respectively. The cells were seeded at a density of 400 to 800 cells/well in 384-well plates and then incubated with the compounds in a gradient concentration (50.0, 10.0, 2.0, 0.4, and 0.08 µM) or with a solvent control for 72 h, followed by the addition of CCK-8 reagent. The OD value of each well was measured at 450 nm using a SpectraMax M5 Microplate Reader (Molecular Devices). Sorafenib functioned as the positive control. Dose-response curves were plotted to determine IC 50 based upon the average values of three parallel experiments using Prism 5.0.

Molecular Docking Analysis
The molecular docking analysis with the structure of AChE (PDB code: 2CMF) [16] was conducted according to the procedure described previously [20].

Conclusions
In summary, the chemical investigation of the soft coral-derived fungus Aspergillus sclerotiorum SCSIO 41031 has led to four new compounds-three cyclic hexapeptides (1-3), and a new lipodepsipeptide (4). Their structures, including their absolute configurations, were determined by comprehensive spectroscopic methods and X-ray crystallographic analysis together with Marfey's method. The folding of the peptide backbone remains to be studied. The in vitro bioassay and in silico docking study revealed compound 4 to be a potential anti-nasopharyngeal cancer drug and a moderate AChE inhibitor.