Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

Synthetic analogues of D-GlcN alpha 1-6D-myo-inositol-1-HPO4-3(sn-1,2-diacy lglycerol) (GlcN-PI), with the 2-position of the inositol residue substitut ed with an O-octyl ether [D-GlcN alpha 1-6D-(2-O-octyl)myo-inositol-1-HPO4- 3-sn-1,2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D- GlcN alpha 1-6D-(2-O-hexadecyl)myo-inositol-1-HPO4-3-sn-1,2-dipalmitoylglyc erol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of g lycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free sys tems of the protozoan parasite Trypanosoma brucei (the causative agent of h uman African sleeping sickness) and human HeLa cells. Neither these compoun ds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosy nthetic enzymes in the HeLa cell-free system but are potent inhibitors of G PI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI wa s shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrat e for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like Glc N-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl )PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, t he addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generatio n of in vitro parasite GPI pathway-specific inhibitors.