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

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 <i>Aspergillus templicola</i>. This led to the targeted isolation of an unprecedented cyclotetrapeptide, templicolamide A (<b>1</b>, featuring a rare β-enamino acid), and four piperazine derivatives (<b>2</b>-<b>5</b>), including the new helvamide E (<b>2</b>) with a unique bicyclic scaffold. The structures were elucidated through extensive spectroscopic analysis and comparison with literature data, with the absolute configuration of compound <b>1</b> further confirmed by advanced Marfey's method and ECD calculations and that of compound <b>2</b> confirmed by single-crystal X-ray diffraction. All isolates were evaluated for their antipaclitaxel (PTX) resistance activity in <i>P</i>-glycoprotein (P-gp)-overexpressing tumor cell lines. Notably, the piperazine derivatives, particularly compound <b>3</b> (a known structure), exhibited potent MDR reversal. Mechanistic studies demonstrated that <b>3</b> reversed MDR primarily through direct binding to P-gp and inhibiting its efflux function, without affecting its expression. <i>In vivo</i>, the PTX + <b>3</b> 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.