Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Probing the Apoptolidin Target


By: Carol A. Rouzer, VICB Communications
Published: December 9, 2014



Synthesis of fluorescent apoptolidin probes provides important tools for exploring their cellular target and mechanism of selective toxicity for cancer cells.


Apoptolidins are natural products of the soil actinomycete Nocardiopsis sp. FU40. These complex macrocyclic molecules, which are biosynthesized through the polyketide pathway, are cytotoxic to some cancer cells, but not their normal counterparts. The mechanism for this selective cytotoxicity is induction of apoptosis via the mitochondrial pathway, and some evidence suggests that the target of the apoptolidins is the mitochondrial F0F1 ATPase. However, the potency of the apoptolidins for F0F1 ATPase inhibition is considerably lower than their potency for cytotoxicity in cancer cells, leading Vanderbilt Institute of Chemical Biology investigators Gary Sulikowski, Brian Bachmann, Larry Marnett, and Dave Piston to embark on a search for alternative targets. Now, they report the synthesis of fluorescent apoptolidin probes designed to facilitate that search [S.M. DeGuire, et al. (2014) Angew. Chem. Int. Ed., published online November 27, DOI: 10.1002/anie.201408906].

Since the original isolation of apoptolidin A (Figure 1), natural and synthetic variants of the compounds have been discovered. Structure-activity relationships among these compounds indicate that alterations of the macrocyclic core, including deoxygenation, demethylation, and double bond isomerization, have little effect on cytotoxicity. However, removal of all three of the deoxy sugars bound at carbons 9 and 27 of the apoptolidin core produces inactive apoptolidinones such as apoptolidinones A and D, shown in Figure 1.




Figure 1. Structures of various apoptolidins. The macrocyclic core is shown in blue, and the three deoxysugars are shown in red. Apoptolidin A was the first compound isolated from Nocardiopsis sp.FU40. Removal of the disaccharide at C27 produces apoptolidin H, which retains some cytotoxic activity. Removal of the monosaccharide at C9 and the disaccharide at C27 produces the inactive apoptolidinones A and D. However, apoptolidinone D disaccharide, which retains the deoxysugars at C27 is cytotoxic. The EC50 values for cytotoxicity against H292 human lung cancer cells are provided for each compound.


To better evaluate the cytotoxicity of the apoptolidins, the researchers developed an assay using H292 human lung cancer cells. They found that apoptolidin A blocks growth of these cells when they are cultured at low confluency (~20%). However, the compound is highly toxic against high (~70%) confluency cultures, displaying a EC50 (concentration that kills 50% of the cells) of 20 to 30 nm. The investigators hypothesized that cultures grown at low confluency primarily use glycolysis for respiration, whereas cells grown at high confluency rely more on oxidative phosphorylation. Prior work had shown that reliance on glycolysis for energy protects cells from the cytotoxicity of apoptolidins. The assay also revealed that apoptolidin A was even more toxic to cells grown in medium containing low concentrations of glucose, a condition that mimics the low availability of nutrients resulting from poor circulation that is often observed in tumors.


The H292 cell assay demonstrated that compounds lacking some of apoptolidin A’s deoxysugar residues retained cytotoxic activity. For example, apoptolidin H, which lacks the disaccharide at carbon 27, and apoptolidinone D disaccharide, which lacks the monosaccharide at carbon 9, exhibited EC50 values of 810 and 200 nm, respectively. These findings suggested that some structural modifications of the sugar moieties might be tolerated. The investigators exploited this discovery by adding a 5-azidopentanoic acid moiety to the alchohol at carbon-2′ of apoptolidins A and H (Figure 2). The modified compounds retained cytotoxic activities comparable to those of their parent molecules, and the azido group provided a site for attachment of a fluorescent tag using strain-promoted alkyne-azido cycloaddition chemistry. For this purpose, the researchers used bicycle[6.1.0]nonynes linked to the fluorescent dye Cy-3 or to biotin (BNE-Cy-3 and BNE-biotin, Figure 2) as the alkynes to react with the azido apoptolidins. This approach yielded Cy-3 apoptolidin A, Cy-3 apoptolidin H, and biotin apoptolidin A, all of which retained cytotoxic activity (Figure 2).




Figure 2. Structures of synthetic apoptolidin probes. Addition of 5-azidopentanoic acid (green) at carbon-2′ of apoptolidins A and H yields the cytotoxic azido apoptolidins A and H (top). A strain-promoted alkyne-azido cycloaddition reaction was then used to couple BNE-Cy-3 or BNE-biotin (blue and red, center) to the azide functional group of the apoptolidins. The products, Cy-3 apoptolidins A and H and biotin apoptolidin A (bottom) were all cytotoxic. The EC50 values are provided for each of the apoptolidins in the H292 lung carcinoma cell assay.


Incubation of H292 cells with either Cy-3 apoptolidin A or Cy-3 apoptolidin H led to fluorescent staining of the cells. In both cases, the dye colocalized with Mitotracker Green FM, suggesting that the apoptolidins were localized to the mitochondria (Figure 3). The authors note that cationic dyes, such as Cy-3, also localize in the mitochondria, so further work will be required to determine if the localization of the apoptolidin probes truly reflects targeting that is mediated by the natural products. However, this research clearly demonstrates a successful approach for the synthesis of cytotoxic apoptolidin probes. Such probes will serve as valuable tools to explore the role of the sugar moieties in apoptolidin targeting and to identify the actual target, a critical step towards understanding the mechanism of action of these very interesting compounds.



Figure 3. Staining of H292 cells with Cy-3 apoptolidin A (left) or Cy-3 apoptolidin H (right). Each group of four micrographs shows staining with the fluorescent apoptolidin (top left), staining with Mitotracker Green FM (top right), differential interference microscopy image (bottom left), and merged fluorescence of the apoptolidin and Mitotracker Green FM. Figure kindly provided by Gary Sulikowski, copyright 2014..







The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center

  • Areas of Interest