Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

Synthetic analogues of D ‐GlcNα1–6 D ‐ myo ‐inositol‐1‐HPO 4 ‐3(sn‐1,2‐diacylglycerol) (GlcN‐PI), with the 2‐position of the inositol residue substituted with an O ‐octyl ether [D‐GlcNα1–6 D ‐(2‐ O ‐octyl) myo ‐inositol‐1‐HPO 4 ‐3‐sn‐1,2‐dipalmitoylglycerol; GlcN‐(2‐ O ‐octyl) PI] or O ‐hexadecyl ether [D‐GlcNα1–6 D ‐(2‐ O ‐hexadecyl) myo ‐inositol‐1‐HPO 4 ‐3‐sn‐1,2‐dipalmitoylglycerol; GlcN‐(2‐ O ‐hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell‐free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N ‐acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell‐free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell‐free system. GlcN‐(2‐ O ‐hexadecyl)PI was shown to inhibit the first α‐mannosyltransferase of the trypanosomal GPI pathway. The N‐acetylated derivative GlcNAc‐(2‐ O ‐octyl)PI is a substrate for the trypanosomal GlcNAc‐PI de‐ N ‐acetylase and this compound, like GlcN‐(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, the addition of the ethanolamine phosphate bridge in the T.brucei cell‐free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway‐specific inhibitors.