Molecular Networking-Driven Discovery of an Unprecedented Cyclotetrapeptide and Potent Anti-MDR P-Glycoprotein Modulating Piperazines from Aspergillus templicola

Multidrug resistance (MDR) remains a significant challenge in cancer chemotherapy. Seeking novel MDR modulators, we employed a molecular networking (MN)-guided strategy to explore the endophytic fungus Aspergillus templicola. This led to the targeted isolation of an unprecedented cyclotetrapeptide, templicolamide A (1, featuring a rare β-enamino acid), and four piperazine derivatives (2–5), including the new helvamide E (2) with a unique bicyclic scaffold. The structures were elucidated through extensive spectroscopic analysis and comparison with literature data, with the absolute configuration of compound 1 further confirmed by advanced Marfey’s method and ECD calculations and that of compound 2 confirmed by single-crystal X-ray diffraction. All isolates were evaluated for their antipaclitaxel (PTX) resistance activity in P-glycoprotein (P-gp)-overexpressing tumor cell lines. Notably, the piperazine derivatives, particularly compound 3 (a known structure), exhibited potent MDR reversal. Mechanistic studies demonstrated that 3 reversed MDR primarily through direct binding to P-gp and inhibiting its efflux function, without affecting its expression. In vivo, the PTX + 3 combination achieved effective tumor regression and apoptosis in a xenograft model with no observable toxicity. Our findings broaden the structural diversity of fungal MDR modulators and underscore the potential of specific nontoxic piperazine scaffolds as promising anti-MDR therapeutic leads.