Structure-activity relationships within a family of selectively cytotoxic macrolide natural products.

[figure: see text] We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity. We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target. Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.

Nicotine and amyloid formation.

The major protein constituents of amyloid deposits in Alzheimer's disease (AD) are the 40-residue beta-amyloid (Abeta) (1-40) peptide and the 42-residue Abeta(1-42) peptide. The Abeta(1-42) is more pathogenic and produced in greater quantities in familial forms of AD. A major goal of research is to uncover a suitable inhibitor that either slows down or inhibits Abeta formation (beta-amyloidosis). During beta-amyloidosis, structural changes associated with the conversion of monomeric Abeta peptide building blocks into the aggregated fibrillar beta-sheet structures occur (alpha-helix-->beta-sheet or random, extended chain-->beta-sheet). In previous work, we and others established that nicotine, a major component of cigarette smoke, inhibits beta-amyloidosis of the Abeta(1-42), which may result from nicotine binding to the alpha-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Abeta(1-28). This information suggests that, when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition. In this report, NMR studies were augmented with the naturally occurring Abeta(1-42), under conditions where the peptide folds into a predominantly alpha-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition to beta-amyloidosis probably results from binding to a small, soluble beta-sheet aggregate that is NMR invisible.

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

BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor 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-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase. SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F(0)F(1)-ATPase.

Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.

Nicotine inhibits amyloid formation by the beta-peptide.

The 42-residues beta-(1-42) peptide is the major protein component of amyloid plaque cores in Alzheimer's disease. In aqueous solution at physiological pH, the synthetic beta-(1-42) peptide readily aggregates and precipitates as oligomeric beta-sheet structures, a process that occurs during amyloid formation in Alzheimer's disease. Using circular dichroism (CD) and ultraviolet spectroscopic techniques, we show that nicotine, a major component in cigarette smoke, inhibits amyloid formation by the beta-(1-42) peptide. The related compound cotinine, the major metabolite of nicotine in humans, also slows down amyloid formation, but to a lesser extent than nicotine. In contrast, control substances pyridine and N-methylpyrrolidine accelerate the aggregation process. Nuclear magnetic resonance (NMR) studies demonstrate that nicotine binds to the 1-28 peptide region when folded in an alpha-helical conformation. On the basis of chemical shift data, the binding primarily involves the N-CH3 and 5'CH2 pyrrolidine moieties of nicotine and the histidine residues of the peptide. The binding is in fast exchange, as shown by single averaged NMR peaks and the lack of nuclear Overhauser enhancement data between nicotine and the peptide in two-dimensional NOESY spectra. A mechanism is proposed, whereby nicotine retards amyloidosis by preventing an alpha-helix-->beta-sheet conformational transformation that is important in the pathogenesis of Alzheimer's disease.