Fluorescent Probes of the Apoptolidins and their Utility in Cellular Localization Studies

Abstract

Apoptolidin A has been described as among the top 0.1% most cell selective cytotoxic agents to be evaluated in the NCI 60 cell line panel. The molecular structure of apoptolidin A consists of a 20-membered macrolide with mono-and disaccharide moieties located at C9 and C27, respectively. In contrast to apoptolidin A, the aglycone (apoptolidinone) shows no cytotoxicity (>10 μM) when evaluated against several tumor cell lines. Apoptolidin H, the C27 deglycosylated analog of apoptolidin A, was produced by targeted glycosyl transferase gene deletion and displayed sub-micromolar activity against H292 lung carcinoma cells. Selective esterification of the C2′ hydroxyl group of apoptolidins A and H with 5-azidopentanoic acid afforded azido functionalized derivatives of potency equal to their parent macrolide. Azido apoptolidins readily underwent strain-promoted alkyne azido cycloaddition (SPAAC) reactions to provide access to fluorescent and biotin functionalized probes. Microscopy studies demonstrate apoptolidins A and H localize in the mitochondria of H292 human lung carcinoma cells.Keywords natural products; antitumor; polyketides; metabolism; chemical probeThe apoptolidins are macrocylic natural products produced by an actinomycete (Nocardiopsis sp. FU40) soil microbe by way of a type I polyketide synthase biosynthetic pathway. [1] Apoptolidin A (1) was reported to induce cell death in E1A transformed rat glia cells, a model cancer cell phenotype, while not affecting the growth of non-transformed glia cells. [2] The described selective cytotoxicity of apoptolidin A stimulated interest in its total synthesis and mechanism of induced cell death. [3] Salomon and Khosla employed a pharmacological approach to define the mechanism of cell death using LYas mouse lymphoma cells and concluded cell death proceeded by way of the mitochondria mediated apoptotic pathway (intrinsic pathway). Following the reported isolation of apoptolidin A (1), other structural variants have been described either as minor microbial metabolites, [5] products of isomerization [6] or semi- [7] and total synthesis [8] . When evaluated for cytotoxicity against tumor cells, these apoptolidins reveal considerable tolerance of structural modifications within the macrolide core including deoxygenation [apoptolidins B and C] In contrast to structural changes within the core macrolides, removal of the deoxy sugars resulted in complete loss of activity with EC 50 values of apoptolidinone A (4) and C (5) reported to be greater then 10 μmol against several tumor cell lines in cell viability assays. [8c,9] The observed loss in activity upon exhausitive deglycosylation of the core macrolide presented an opportunity to develop a series of apoptolidin derived probes to support mechanism of action studies. We report here methods to access apoptolidins of varying state of glycosylation (tri-, di-, mono and non-glycosylated) and preliminary studies on their use as cellular probes.Apoptolidin A (1) is readily obtained by fermentation of the actinomyce...

Novel fluorescent probes of 10-hydroxyevodiamine: autophagy and apoptosis-inducing anticancer mechanisms

“…The chemical syntheses of the probes are depicted in Scheme 1, Scheme 2. Compound 1 was prepared according to our reported procedure12., 13.. Then, di- tert -butyl dicarbonate was used to protect 6-aminohexanoic acid ( 2a ) to afford compound 3a , which was subsequently reacted with compound 1 in the presence of EDC and DMAP to give compound 4a .…”

“…In particular, a series of novel evodiamine derivatives were prepared that showed excellent in vitro and in vivo antitumor activity and were promising lead compounds for the development of novel antitumor agents 12. , 13. .…”

“…Elucidating the mechanisms of bioactive NPs is challenging because of the limitations of analytical approaches 8 . Recently, fluorescent probes, which often act as a valuable tool to investigate the distribution of compounds in cells9., 10., 11., have been widely used in chemical biology studies of NPs12., 13., 14..…”

“…Previously, our group designed a number of evodiamine derivatives and clarified the structure—activity relationships (SARs)15., 16.. In particular, a series of novel evodiamine derivatives were prepared that showed excellent in vitro and in vivo antitumor activity and were promising lead compounds for the development of novel antitumor agents12., 13.. Importantly, in silico target prediction in combination with antitumor mechanism studies indicated that evodiamine derivatives were dual topoisomerase 1 (Top1) and topoisomerase 2 (Top2) inhibitors and thio-evodiamine derivatives were triple Top1/Top2/tubulin inhibitors 17 .…”

Engineered Biosynthesis of Alkyne-Tagged Polyketides by Type I PKSs

“…One such technology is to tag polyketides with a clickable functionality, which has been demonstrated to facilitate the study of polyketide biosynthesis, biology, and pharmacology through bio-orthogonal chemistry (DeGuire et al, 2015;Harvey et al, 2012;Hughes et al, 2014;Kalkreuter et al, 2019aKalkreuter et al, , 2019bKoryakina et al, 2017;Musiol-Kroll et al, 2017;Riva et al, 2014;Seidel et al, 2019;Zhu and Zhang, 2015). In particular, polyketides can be tagged through semi-synthesis (DeGuire et al, 2015;Seidel et al, 2019), total synthesis (Staub and Sieber, 2008), precursor-directed biosynthesis (Harvey et al, 2012;Koryakina et al, 2017;Musiol-Kroll et al, 2017;Seidel et al, 2017;Yan et al, 2013), or de novo biosynthesis (Zhu et al, 2015a;Zhu and Zhang, 2015). In this work we aim to further develop the strategy of de novo biosynthesis, which offers the unique advantage of not feeding the biorthogonal moiety itself, which could lead to increased background due to the diffusible non-specific nature of feeding starter or extender units.…”