Target Identification of Yaku'amide B and Its Two Distinct Activities against Mitochondrial F_oF₁-ATP Synthase

Yaku'amide B (1b) is a structurally unique tetradecapeptide bearing four β,β-dialkylated α,β-unsaturated amino acid residues. Growth-inhibitory profile of 1b against a panel of 39 human cancer cell lines is distinct from those of clinically used anticancer drugs, suggesting a novel mechanism of action. We achieved total syntheses of chemical probes based on 1b and elucidated the cellular target and mode of action of 1b. Fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b were prepared for cell imaging studies and pull-down assays, respectively. In addition, the unnatural enantiomer of 1b ( ent-1b) and its fluorescent probe ( ent-3) were synthesized for control experiments. Subcellular localization analysis using 3 and 4 showed that 1b selectively accumulates in the mitochondria of MCF-7 human breast cancer cells. Pull-down assays with 6 revealed F_oF₁-ATP synthase as the major target protein of 1b. Consistent with these findings, biochemical activity assays showed that 1b inhibits ATP production catalyzed by mitochondrial F_oF₁-ATP synthase. Remarkably, 1b was also found capable of enhancing the ATP hydrolytic activity of F_oF₁-ATP synthase. On the other hand, ent-1b inhibits ATP synthesis more weakly than does 1b and does not affect ATP hydrolysis, suggesting the stereospecific requirement for the characteristic multimodal functions of 1b. These findings corroborate that 1b causes growth arrest in MCF-7 cells by inhibiting ATP production and enhancing ATP hydrolysis, thereby depleting the cellular ATP pool. This study provides, for the first time, a structural basis for the design and development of anticancer agents exploiting the novel mode of action of 1b.