Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis.
Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40-50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.
Mellor
Howard R
HR
Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Neville
David C A
DC
Harvey
David J
DJ
Platt
Frances M
FM
Dwek
Raymond A
RA
Butters
Terry D
TD
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D002250:Carbon Radioisotopes / Q000378:metabolism; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006028:Glycosphingolipids / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000378:metabolism; D018922:HL-60 Cells / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D006028:Glycosphingolipids; D017485:1-Deoxynojirimycin