Tailoring structure–function and targeting properties of ceramides by site-specific cationization

In the course of our studies on compartment-specific lipid-mediated cell regulation, we identified an intimate connection between ceramides (Cers) and the mitochondria-dependent death-signaling pathways. Here, we report on a new class of cationic Cer mimics, dubbed ceramidoids, designed to act as organelle-targeted sphingolipids (SPLs), based on conjugates of Cer and dihydroceramide (dhCer) with pyridinium salts (CCPS and dhCCPS, respectively). Ceramidoids having the pyridinium salt unit (PSU) placed internally (α and γ- CCPS) or as a tether (ω-CCPS) in the N-acyl moiety were prepared by N-acylation of sphingoid bases with different ω-bromo acids or pyridine carboxylic acid chlorides following capping with respective pyridines or alkyl bromides. Consistent with their design, these analogs, showed a significantly improved solubility in water, well-resolved NMR spectra in D2O, broadly modified hydrophobicity, fast cellular uptake, and higher anticancer activities in cells in comparison to uncharged counterparts. Structure–activity relationship (SAR) studies in MCF-7 breast carcinoma cells revealed that the location of the PSU and its overall chain length affected markedly the cytotoxic effects of these ceramidoids. All ω-CCPSs were more potent (IC50/48 h: 0.6–8.0 μM) than their α/γ-CCPS (IC50/48 h: 8–20 μM) or D-erythro-C6-Cer (IC50/48 h: 15 μM) analogs. ω-DhCCPSs were also moderately potent (IC50/48 h: 2.5–12.5 μM). Long-chain ω-dhCCPSs were rapidly and efficiently oxidized in cells to the corresponding ω-CCPSs, as established by LC–MS analysis. CCPS analogs also induced acute changes in the levels and composition of endogenous Cers (upregulation of C16-, C14-, and C18-Cers, and downregulation of C24:0- and C24:1-Cers). These novel ceramidoids illustrate the feasibility of compartment-targeted lipids, and they should be useful in cell-based studies as well as potential novel therapeutics.

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