C-Terminal modification of polytheonamide B uncouples its dual functions in MCF-7 cancer cells.

Polytheonamide B (1) is an exceptionally large peptide that forms a transmembrane ion channel. The potent cytotoxicity of 1 against MCF-7 cancer cells originates from its two ion transport functions. Compound 1 depolarizes the plasma membrane and neutralizes acidic lysosomes. Here, we describe how we uncoupled these functions by designing and synthesizing new analogues of 1.

Gramicidin A accumulates in mitochondria, reduces ATP levels, induces mitophagy, and inhibits cancer cell growth.

Gramicidin A (1) is a linear 15-mer peptidic natural product. Because of its sequence of alternating d- and l-chirality, 1 folds into a ß6.3-helix in a lipid bilayer and forms a head-to-head dimer to function as a transmembrane channel for monovalent cations (H+, Na+, and K+). The potent anticancer activity of 1 was believed to be mainly attributed to the free ion diffusion across the plasma membrane. In this study, we investigated the cytostatic action of 1 in nanomolar concentrations using the human breast cancer cell line MCF-7, and revealed the unprecedented spatiotemporal behavior of 1 for the first time. Compound 1 not only disrupted the ion concentration gradients of the plasma membrane, but also localized in the mitochondria and depolarized the inner mitochondrial membrane. The diminished H+ gradient in the mitochondria inhibited ATP synthesis. The resultant mitochondrial malfunction led to mitophagy, while the cellular energy depletion induced G1 phase accumulation. The multiple events occurred in a time-dependent fashion and ultimately caused potent inhibition of cell growth. The present study provides valuable information for the design and development of new cytostatic agents exploiting channel-forming natural products.

Biological Effects of a Simplified Synthetic Analogue of Ion-Channel-Forming Polytheonamide†B on Plasma Membrane and Lysosomes.

Polytheonamide B (1) is a linear 48-mer natural peptide with alternating d- and l-amino acid residues. Compound 1 forms conducting channels for monovalent ions and exhibits potent cytotoxicity against MCF-7 cells. Previously, we reported that nanomolar concentrations of 1 induce plasma membrane depolarization and lysosomal pH disruption, which triggers apoptosis. Here, we report the cellular localization and biological action of a simplified synthetic analogue of 1, polytheonamide mimic 3. Compared with 1, the toxicity of 3 against MCF-7 cells is 16 times weaker. Although its plasma membrane depolarization effect is only 3.6 times lower, more 3 (20-fold) is required to neutralize lysosomal pH. Thus, the effective concentrations for lysosomal neutralization and cytotoxicity by 3 are comparable. These results strongly suggest that the activity of 3 against the lysosomal membrane is more important for apoptotic cell death than its effects on the plasma membrane, and provide valuable information regarding the unique behavior of polytheonamide-based molecules.