Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness

Abstract

It has been suggested that compounds affecting glycosylphosphatidylinositol (GPI) biosynthesis in bloodstream form Trypanosoma brucei should be trypanocidal. We describe cell-permeable analogues of a GPI intermediate that are toxic to this parasite but not to human cells. These analogues are metabolized by the T. brucei GPI pathway, but not by the human pathway. Closely related nonmetabolizable analogues have no trypanocidal activity. This represents the first direct chemical validation of the GPI biosynthetic pathway as a drug target against African human sleeping sickness. The results should stimulate further inhibitor design and synthesis and encourage the search for inhibitors in natural product and synthetic compound libraries.

Figure 1

Novel analogues of GlcN-PI and their metabolism by a trypanosome cell-free system. (A) Structures of the synthetic GlcN-PI analogues used in this study. (B) Synthetic GlcNAc-PI and GlcN-PI (positive controls, lanes 2 and 3) and analogues thereof (compounds 1–6, lanes 4–9) were incubated with a modified trypanosome cell-free system and GDP-[³H]Man. Radiolabelled glycolipid products were analysed by HPTLC and fluorography. The products were characterized by enzymatic and chemical digestions (Table I) and are: DPM, dolichol-P-mannose; M1-3, Man_1–3GlcNα1-6PI; aM3, Man₃GlcNα1-6(2-O-acyl)PI; A′, EtNP-Man₃GlcNα1-6PI; C′, EtNP-Man₃GlcNα1-6(2-O-acyl)PI; X–Z, Man_1–3GlcNα1-6(2-O-methyl)D-myo-inositol1-P-octadecanol; U–W, Man_1–3GlcNα1-6(2-O-methyl) (?-O-acyl)D-myo-inositol1-P-octadecanol. (C) Synthetic GlcNAc-PI (control, lanes 2 and 3) and compound 3 were incubated with the trypanosome cell-free system in the presence (+) and absence (−) of PMSF. Radiolabelled glycolipid products were analysed by HPTLC and fluorography.

Figure 2

Compound 1 is metabolized by the trypanosome GPI pathway in vivo and inhibits the formation of glycolipid C. (A) Living trypanosomes were pre-incubated with 50 μM compound 1 or compound 5 for 30–60 min, as indicated, and then labelled for 60 min with [³H]Man. Extracted glycolipids were analysed by HPTLC and fluorography. The products, which were characterized by enzymatic and chemical digestions (Table I), are as described in the legend to Figure 1. (B) As for (A), but labelling with [³H]myristate. (C) As for (A), but with a fixed pre-incubation time of 60 min and varying concentrations of compound 1. Compound 1 was added as a butanol solution and a control with butanol alone is included (lane 2). The boxed data (lane 6) are from a cell-free system labelling experiment to provide markers for glycolipids U–Z. (D) Trypanosomes were pre-incubated without any additive (lane 1), or with 30 μM compound 1 (lane 2), 30 μM compound 2 (lane 3) or 60 μg/ml cycloheximide (lane 4) for 60 min, labelled with [³H]myristate for 60 min, lysed in boiling sample buffer and analysed by SDS–PAGE and fluorography. The radiolabel incorporated in the presence of the protein synthesis inhibitor cycloheximide represents that due to myristate exchange on pre-existing VSG rather than de novo synthesis of VSG. (E) Summary of the fates of compounds 1 and 2, as indicated by the in vivo labelling data (see text).

Figure 3

Compound 3 does not directly inhibit the formation of glycolipid C. (A) The trypanosome cell-free system was pulse-labelled with GDP-[³H]Man, to form labelled glycolipids A′ and θ, and then chased with cold GDP-Man and a fatty-acid remodelling mix. The remodelled (glycolipid A) and inositol acylated (glycolipid C) products were extracted and analysed by HPTLC and by scanning with a linear analyser. (B) As for (A), except that PMSF was added to the chase. (C) As for (A), except that compound 3 was added to the chase.

Figure 4

Selective toxicity of a GPI analogue (compound 1) to bloodstream from T. brucei. Parasites were cultured in the presence of 0 (control), 10, 20, 30 and 40 μM compound 1. Viable cells were estimated after 2, 4, 6 and 8 h and normalized to a percentage of the control cultures (values are means of at least nine separate determinations and standard errors are ⩾±5%).