The growing interest in single-molecule analysis of RNA calls for programmable enzymatic labeling strategies beyond the horizon of solid-phase synthesized RNAs. Herein we describe an easy and versatile chemo-enzymatic approach to label RNA at its termini or defined internal positions via click-chemistry.

Induction of apoptosis mediated by the inhibition of ceramidases has been shown to enhance the efficacy of conventional chemotherapy in several cancer models. Among the inhibitors of ceramidases reported in the literature, B-13 is considered as a lead compound having good in vitro potency towards acid ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid ceramidase in living cells, LCL-464 a modified derivative of B-13 containing a basic ω-amino group at the fatty acid was reported to have higher potency towards lysosomal acid ceramidase in living cells. In a search for more potent inhibitors of ceramidases, we have designed a series of compounds with structural modifications of B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endogenous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows that structural modification of the known ceramidase inhibitors B-13 and LCL-464 generates more potent ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but also enhances cell death.In this study we describe the design and synthesis of a series of modified B-13 and LCL-464 analogues with different substituents as inhibitors of recombinant acid and neutral ceramidases. We show that the inhibition potential of B-13 could be enhanced both in in vitro and in intact cells by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed some potent compounds for the inhibition of both aCDase and nCDase with the elevation of ceramide level.

First oligonucleotide analogues that inhibit miRNA function are currently investigated in clinical trials. In addition, several alternative methods are under development that may allow for controlling miRNA function by small molecules-mediated inhibiting of its biogenesis. In this perspectives article, we provide a short overview on recent developments in this field.

Ceramidases are key regulators of cell fate. The biochemistry of different ceramidases and of their substrate ceramide appears to be complex, mainly due to specific biophysical characteristics at the water-membrane interface. In the present study, we describe the design and synthesis of a set of fluorescently labeled ceramides as substrates for acid and neutral ceramidases. For the first time we have replaced the commonly used polar NBD-dye with the lipophilic Nile Red (NR) dye. Analysis of kinetic data reveal that although both the dyes do not have any noticeable preference for the substitution at acyl or sphingosine (Sph) part in ceramide towards hydrolysis by acid ceramidase, the ceramides with acyl-substituted NBD and Sph-substituted NR dyes have been found to be a better substrate for neutral ceramidase.In this study we have synthesized novel Nile Red (NR)-labeled ceramides as substrates of acid and neutral ceramidases. Structure–activity-relationship and kinetic studies with NR- and commercially available NBD-ceramides provide insights into their substrate specificities toward recombinant acid and neutral ceramidases.

In the present study we describe the design and synthesis of a series of amide- and sulfonamide-based compounds as inhibitor of recombinant acid and neutral ceramidases. Inhibition of ceramidases has been shown to induce apoptosis and to increase the efficacy of conventional chemotherapy in several cancer models. B-13, lead in vitro inhibitor of acid ceramidase has been recently shown to be virtually inactive towards lysosomal acid ceramidase in living cells at lower concentrations and for a shorter time of treatment, suggesting the development of more potent inhibitors. In this study, a detailed SAR investigation has been performed to understand the effect of different substituents on the phenyl ring of amide- and sulfonamide-based compounds that partially resemble the structure of well-known inhibitors such as B-13, D-e-MAPP as well as NOE. Our results suggest that the electronic effects of the substituents on phenyl ring in B-13 and D-e-MAPP analogues have negligible effects either in enhancing the inhibition potencies or for selectivity towards aCDase over nCDase. However, the hydrophobicity and the steric effects of longer alkyl chains (n-Pr, n-Bu or t-Bu groups) at the phenyl ring were found to be important for an enhanced selectivity towards aCDase over nCDase.In the present study we describe the design and synthesis of series of amide- and sulfonamide-based compounds as inhibitors of recombinant acid and neutral ceramidases. Our results suggest that the electronic effects of the substituents on phenyl ring in B-13 and D-e-MAPP analogues have negligible effects either in enhancing the inhibition potencies or for selectivity towards aCDase over nCDase. However, the hydrophobicity and the steric effects of longer alkyl chains (n-Pr, n-Bu or t-Bu groups) at the phenyl ring were found to be important for an enhanced selectivity towards aCDase over nCDase.

The acid sphingomyelinase is an emerging drug target, especially for inflammatory lung diseases. Presently, there are no directly-acting potent inhibitors available for cell-based studies. The potent inhibitor phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2) is not only unsuited for cell culture studies, but also does not provide hints for further structural improvements. In the SAR study described here, we replaced the inositolphosphate moiety by a carbohydrate derivative and the phosphatidic acid residue by an alkylsulfone ester. The resulting compound is more active than its parent compound and offers new means for further structural modification.A carbohydrate-derived analogue of potent and selective acid sphingomyelinase inhibitor phosphatidylinositol-3,5-bisphosphate was synthesized. This compound is more potent and as selective as its parent compound.

•Fluorescent sphingolipids are perfect for FRET studies in lipid–protein interaction.•Biotin-GM1 discloses intracellular localization by immuno-electron microscopy.•Photoactivatable GM2 labels the binding site of GM2 activator protein.•Non-degradable glycosphingolipids for metabolic and traffic studies•Synthetic inhibitors of acid sphingomyelinaseThe unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.

The growing interest in single-molecule analysis of RNA calls for programmable enzymatic labeling strategies beyond the horizon of solid-phase synthesized RNAs. Herein we describe an easy and versatile chemo-enzymatic approach to label RNA at its termini or defined internal positions via click-chemistry.