Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase

Background: Apoptolidin is a macrolide originally identified on the basis o f its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study w as to identify the molecular target of this newly discovered natural produc t, Results: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays a s well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptol idin was independent of p53 status, inhibited by BCL-2, and dependent on th e action of caspase-9. PARP was completely cleaved in the presence of 1 muM apoptolidin within 6 h in a mouse lymphoma cell line. Together these resul ts suggested that apoptolidin might target a mitochondrial protein. Structu ral comparisons between apoptolidin and other macrolides revealed significa nt similarity between the apoptolidin aglycone and oligomycin, a known inhi bitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-1 00-solubilized ATPase preparations. The K-i For apoptolidin was 4-5 muM. Th e selectivity of apoptolidin in the NCI-60 cell line panel was found to cor relate well with that of several known anti-fungal natural products that in hibit the eukaryotic mitochondrial F0F1-ATP synthase. Significance: Although the anti-fungal activities of macrolide inhibitors o f the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cyt ovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin. foll owed by the demonstration that it is an inhibitor of the mitochondrial FI,F I-ATP synthase, highlights the potential relevance of these natural product s as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is disc ussed. (C) 2001 Elsevier Science Ltd. All rights reserved.