Secondary metabolites of selected helicosporous tubeufiaceae

Abstract

Helicosporous hyphomycetes is a group of saprophytic fungi, with occasional endophytic forms. Its characteristic feature is the presence of helicoid conidia, which can coil at least 180° on a two-dimensional or three-dimensional plane. Despite their distinctive morphology and strong biosynthetic potential—yielding alkaloids, glycolipids, polyketides, and terpenoids with antibacterial, antitumor, and antidiabetic activities—chemical studies remain limited. In our prior work, we accumulated an extensive Tubeufiaceae strain collection and, via small-scale solid-state fermentations, generated crude extracts for chemical and cytotoxicity screening. Five strains—Neohelicosporium guangxiense (SLGY-5), N. griseum (GYSLGY-1, SLGY-15), Tubeufia longihelicospora (WZS71), and Helicosporium sexuale (LZ15)—were prioritized for detailed metabolite and bioactivity studies. From Neohelicosporium guangxiense SLGY-5, three α-tetralone derivatives (C1–C3) and one isocoumarin (C4) were isolated; notably, C1 was obtained from natural sources for the first time. From N. griseum GYSLGY-1, two new polyketides—neogrisphenol A (C5) and neogrisphenol B (C6)—one new isochroman-1-one, (S)-6-hydroxy-7-methoxy-3,5-dimethylisochroman-1-one (C7), and four known compounds (C8–C11) were characterized. From N. griseum SLGY-15, seven polyketides (C12–C18) and one steroid (C19) were obtained. From Tubeufia longihelicospora WZS71, two dimeric naphtho-γ-pyrones (C20–C21), two dimeric coumarins (C22–C23), and one alkaloid (C24) were isolated. From Helicosporium sexuale LZ15, seven new dibenzo-α-pyrone derivatives—Helicolide A (C25), Helicohydrins B–D (C26–C28), and Helicochlorins E–G (C29–C31)—together with eight known congeners (C32–C39) were obtained. Structures were elucidated by 1D/2D NMR, mass spectrometry, NOE analysis, single-crystal X-ray diffraction, and ECD, supported by quantum-chemical calculations. Cytotoxicity testing revealed that neogrisphenol A (C5) showed potent activity against A2780, PC-3, and MDA-MB-231 cells (IC₅₀ = 3.20, 10.68, and 16.30 μM, respectively), exceeding the potency of cisplatin (CDDP) in A2780 under the same conditions. Neogrisphenol B (C6) was also active against A2780 (IC₅₀ = 10.13 μM). In vitro mechanistic assays indicated that C5 suppressed A2780 proliferation, induced apoptosis, and caused S-phase cell-cycle arrest in a concentration-dependent manner. Compounds C12, C14, and C15 exhibited cytotoxicity toward HeLa cells (IC₅₀ = 30.8, 13.7, and 14.1 μM) and A549 cells (IC₅₀ = 24.7, 7.4, and 10.3 μM), respectively. Compound C36 exhibited potent activity in HepG2/HeLa (IC₅₀ both < 20 μM), indicating selectivity toward these two cell lines; compound C38 showed moderate activity in A549/HeLa (16.55–26.49 μM), also displaying selectivity; compound C37 was moderate and selective toward A2780 (25.73 μM); and compound C30 showed moderate activity only in A549 (38.81 μM). Compound C32 exhibited inhibitory activity across multiple cell lines with IC₅₀ values of 22.49–85.14 μM, indicating broad-spectrum activity. Antimicrobial testing showed that compounds C5–C6 and C10 were active against Bacillus subtilis, Clostridium perfringens, and Staphylococcus aureus (MIC 16–31 μg/mL). Compounds C12–C15 displayed moderate activity against Pseudomonas aeruginosa, with C13 also showing weak activity against S. aureus. Compounds C20 and C21 were active against P. aeruginosa, with MIC 62.0 μg/mL and MBC 248 μg/mL. In addition, compound C9 showed antifungal activity against Sclerotinia sclerotiorum and Phytophthora nicotianae var. nicotianae, with respective IC50 values of 88.14 ± 2.21 µg/mL and 52.36 ± 1.38 µg/mL. Overall, this work expands the chemical space of helicosporous hyphomycetes and yields promising lead scaffolds; coupling genome mining and metabolomics with SAR/MoA studies around C5, and leveraging scale-up fermentation and semisynthesis for supply and derivatization, will accelerate the discovery of antibacterial and anticancer candidates.