In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by 13 C5 -sphingosine precursor feeding and mass spectrometry.

Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13 C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope-labelled precursor combined with liquid chromatography-mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.

Human glucocerebrosidase mediates formation of xylosyl-cholesterol by ß-xylosidase and transxylosidase reactions.

Deficiency of glucocerebrosidase (GBA), a lysosomal ß-glucosidase, causes Gaucher disease. The enzyme hydrolyzes ß-glucosidic substrates and transglucosylates cholesterol to cholesterol-ß-glucoside. Here we show that recombinant human GBA also cleaves ß-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced ß-glucosidase activity were similarly impaired in ß-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous ß-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing ß-glucosidase GBA2. We later sought an endogenous ß-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived ß-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

Binding kinetics of ZM241385 derivatives at the human adenosine A2A receptor.

Classical drug design and development rely mostly on affinity- or potency-driven structure-activity relationships (SAR). Thus far, a given compound's binding kinetics have been largely ignored, the importance of which is now being increasingly recognized. In the present study, we performed an extensive structure-kinetics relationship (SKR) study in addition to a traditional SAR analysis at the adenosine A2A receptor (A2A R). The ensemble of 24 A2A R compounds, all triazolotriazine derivatives resembling the prototypic antagonist ZM241385 (4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenol), displayed only minor differences in affinity, although they varied substantially in their dissociation rates from the receptor. We believe that such a combination of SKR and SAR analyses, as we have done with the A2A R, will have general importance for the superfamily of G protein-coupled receptors, as it can serve as a new strategy to tailor the interaction between ligand and receptor.