A nonadride derivative from the marine-derived fungus Aspergillus chevalieri PSU-AMF79

Abstract One new nonadride enantiomer, ent-epiheveadride, along with five known dioxopiperazine derivatives were isolated from the marine-derived fungus Aspergillus chevalieri PSU-AMF79. Their structures were identified by extensive spectroscopic analysis. The absolute configuration of ent-epiheveadride was determined by comparison of the specific rotation and electronic circular dichroism data with those of related known compounds. It exhibited antifungal activity against Cryptococcus neoformans ATCC90113 flucytosine–resistant and Candida albicans NCPF3153 with the MIC values of 128 and 200 µg/mL, respectively. In addition, the known L-alanyl-L-tryptophan anhydride displayed TMEM16A inhibitory activity with 65.0% inhibition at a concentration of 5 µg/mL. Graphical Abstract


Introduction
Aspergillus is widely known to be a rich source of secondary metabolites with promising potential as drug candidates (Frisvad and Larsen 2015). The fungus Aspergillus chevalieri, which is also known by its teleomorph named Eurotium chevalieri, is one of the most frequently isolated Aspergillus species (Siqueira et al. 2018). Some bioactive terpene (Ellestad et al. 1972), anthraquinone (Bachmann et al. 1979), hydroquinone (Hamasaki et al. 1981) and dioxopiperazine (Greco et al. 2015) derivatives were successfully isolated from this fungus. However, the report on secondary metabolites from the marine derived fungus A. chevalieri is very limited. Recently, the mutual biotic stress between A. chevalieri and Talaromyces calidicanius significantly enhanced the biosynthesis of acetylcholinesterase inhibitors in comparison with the individual cultures (de Oliveira et al. 2021). During our ongoing effort to investigate the novel bioactive substances from marine-derived fungi in Thailand, Aspergillus chevalieri PSU-AMF79, isolated from Phallusia nigra which was collected from Phuket Province, Thailand, was studied. The broth ethyl acetate extract showed antifungal activity against Cryptococcus neoformans 90113 with an MIC value of 32 mg/mL. Herein, we report the isolation, structural elucidation as well as antimicrobial and TMEM16A inhibitory activities of secondary metabolites from the marine-derived fungus A. chevalieri PSU-AMF79.
Evaluation of antimicrobial activities of all isolated compounds against gram-negative bacteria (Acinetobacter baumannii NPRC005, A. baumannii NPRC007, Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853), gram-positive bacteria (Staphylococcus aureus and methicillin-resistant S. aureus), yeast (Cryptococcus neoformans ATCC90112 flucytosine-resistant, C. neoformans ATCC90113 flucytosine-resistant and Candida albicans NCPF3153) and pathogenic fungi (Microsporum gypseum SK-MU4 and Talaromyces marneffei PSU-SKH1) showed that compound 1 exhibited antifungal activity against C. neoformans ATCC90113 flucytosine-resistant and C. albicans NCPF3153 with the MIC values of 128 and 200 mg/mL, respectively, while compounds 2 and 5 displayed weaker activity against C. neoformans ATCC90113 flucytosine-resistant with equal MIC values of 200 mg/mL. The remaining compounds showed no antimicrobial activity at the concentration of 200 mg/mL. In addition, compounds 2-6 were tested for TMEM16A and CFTR inhibitory activities at the concentration of 5 mg/ mL as well as cytotoxic activity against Calu-3, MCF-7 and HCT-116 cells at the concentration of 10 mg/mL. Only compound 3 exhibited activity on TMEM16A with 65.0% inhibition. None of them was active against all tested cancer cells.

General experimental procedures
Melting points were measured on Electrothermal 9100. The infrared (IR) spectra were recorded using a Perkin-Elmer spectrum BX FT-IR spectrometer. The ultraviolet (UV) absorption spectra were measured in MeOH on a Shimadzu UV-2600 UV-Vis spectrophotometer. Electronic Circular Dichroism (ECD) spectra were recorded on a JASCO J-815 polarimeter. The 1 H and 13 C Nuclear Magnetic Resonance ( 1 H and 13 C NMR) spectra were recorded on 300 MHz Bruker FTNMR Ultra Shield TM spectrometer. The 1D and 2D NMR spectra were recorded using standard Bruker pulse sequences. The specific rotations were recorded on a JASCO P-2000 polarimeter. Mass spectra were obtained on a Bruker MicrOTOF mass spectrometer and Liquid chromatograph-mass spectrometer (2090, LCT, water, micromass). Thin-layer chromatography (TLC) and preparative TLC were carried out on silica gel 60 GF 254 (Merck). Column chromatography (CC) was performed on silica gel (Merck) type 100 (70-230 mesh ASTM) and type 60 (230-400 mesh ASTM) or Sephadex LH-20.

Fungal material
The marine-derived fungus PSU-AMF79 (BCC84321) was isolated from a marine ascidian, Phallusia nigra, collected from Phuket Province, Thailand. This isolate was identified based on its morphological and molecular characteristics. Colonies on potato dextrose agar (PDA) at 25 C are green with diffusible yellowish green pigment. In addition, the molecular analysis of the internal transcribed spacers (ITS) ribosomal DNA revealed that PSU-AMF79 (GenBank accession number MF780705) closely matched with Aspergillus chevalieri KX463363, KX463349 and KX463348 (100% nucleotide identity). Hence, this isolate can be identified as Aspergillus chevalieri.

Antimicrobial assay
Investigation of antimicrobial activity was conducted based on the Clinical and Laboratory Standards Institute (Phongpaichit et al. 2006). The lowest concentration that inhibited visible growth was defined as Minimum Inhibitory Concentration (MIC). Amphotericon B was used as positive control against C. neoformans ATCC90113 flucytosine-resistant and C. albicans NCPF3153 with the MIC values of 1 and 0.25 mg/mL, respectively.

TMEM16A and CFTR inhibitory assays
Both TMEM16A and CFTR chloride channel activities was measured using chamber experiments in Calu-3 and T84 cells, respectively. The TMEM16A inhibitory activity was conducted based on a previous study by Yimnual et al. (2021). Investigation of CFTR inhibitory activity was previously described by Phainuphong et al. (2018).