Anti-colorectal cancer bioactive metabolites from endophytic fungi of Pyrola spp. targeting p-ATM/P53 pathway driven by in vitro experiments and network pharmacology.

Employing the OSMAC (One Strain Many Compounds) strategy, a systematic investigation was conducted on secondary metabolites produced by the co-culture of endophytic fungi Penicillium soppii and Penicillium solitum isolated from Pyrola spp., leading to the isolation of ten compounds from their fermented products. Asperphenamate (5) amid all the compounds exhibited stronger anti-colorectal cancer (CRC) activity against HCT-8 cells than the positive control 5-fluorouracil (P < 0.05), significantly reducing the migration rate of HCT-8 cells in a wound healing assay, increasing the percentage of apoptotic cells from 28.13 ± 0.85 % to 45.58 ± 2.01 % as determined by live/dead cell staining assay, and blocking cell cycle progression from G1 to S phase analyzed by flow cytometry. Asperphenamate (5) may induce apoptosis or cell cycle arrest by activating the DNA damage response (DDR) as evidenced by significant enrichment of DDR-associated signaling pathways and strong binding affinity to key DDR-related proteins through network pharmacology and docking analyses. Furthermore, Asperphenamate (5) increased the expression of p53 and phosphorylated ATM (p-ATM) proteins and decreased CDK2 levels in a concentration-dependent manner, indicating it may be mediated by regulation of the ATM/p53/CDK2 signaling axis, resulting in cell cycle arrest or apoptosis. Collectively, these findings established Asperphenamate (5) as a viable lead compound for CRC therapy and underscored Penicillium spp. as a prolific source of bioactive natural products with medicinal properties.