Discovery of Hirsutellones as Nucleolin-Targeting Natural Macrocycles for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) remains a major clinical challenge due to its aggressive behavior, high metastatic potential, and limited therapeutic options, as current treatments often constrained by resistance and toxicity. Here, ten structurally distinct macrocyclic alkaloids were discovered from Didymella sp. Compounds 1 and 2 possessed the 13-membered macrocyclic ring alongside the novel spiro junction at C-3/C-18. Among them, phomapyrrolidone A (Ppd A) was the most active compound in the series, showing the lowest IC50 values and favorable selectivity toward TNBC cells. Integrated proteomic profiling, bioinformatic analysis, and clinical tissue validation converged on nucleolin (NCL) as a direct and functionally relevant target. Mechanistically, Ppd A binds to the Gln558 residue of NCL, disrupts the NCL-EGFR complex, and suppresses EGFR-PI3K-AKT signaling, leading to apoptosis and tumor growth inhibition. These findings establish NCL as a promising therapeutic target and highlight hirsutellone-type alkaloids as a novel class of NCL modulators for TNBC treatment.