Arthur Crossman

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues

Glycosylphosphatidylinositol (GPI) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. GPI structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. The high density of GPI structures at all life‐cycle stages of African trypanosomes and Leishmania suggests that the GPI biosynthetic pathway might be a reasonable target for the development of anti‐parasite drugs. In this paper we show that synthetic analogues of early GPI intermediates having the 2‐hydroxyl group of the D‐myo‐inositol residue methylated are recognized and mannosylated by the GPI biosynthetic pathways of Trypanosoma brucei and Leishmania major but not by that of human (HeLa) cells. These findings suggest that the discovery and development of specific inhibitors of parasite GPI biosynthesis are attainable goals. Moreover, they demonstrate that inositol acylation is required for mannosylation in the HeLa cell GPI biosynthetic pathway, whereas it is required for ethanolamine phosphate addition in the T.brucei GPI biosynthetic pathway.

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

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.

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

Synthetic analogues of D‐GlcNα1–6D‐myo‐inositol‐1‐HPO4‐3(sn‐1,2‐diacylglycerol) (GlcN‐PI), with the 2‐position of the inositol residue substituted with an O‐octyl ether [D‐GlcNα1–6D‐(2‐O‐octyl)myo‐inositol‐1‐HPO4‐3‐sn‐1,2‐dipalmitoylglycerol; GlcN‐(2‐O‐octyl) PI] or O‐hexadecyl ether [D‐GlcNα1–6D‐(2‐O‐hexadecyl)myo‐inositol‐1‐HPO4‐3‐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 Man2GlcN‐(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 Man1–3GlcN‐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.

Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc‐PI de‐N‐acetylases were determined using 24 substrate analogues. The results show the following. (i) The de‐N‐acetylases show little specificity for the lipid moiety of GlcNAc‐PI. (ii) The 3′‐OH group of the GlcNAc residue is essential for substrate recognition whereas the 6′‐OH group is dispensable and the 4′‐OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing βGlcNAc or aromatic N‐acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR‐PI analogues are de‐N‐acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy‐amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite‐specific suicide substrate inhibitors. These are potential lead compounds for the development of anti‐protozoan parasite drugs.

The N-Acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis Is a Zinc Metalloenzyme

The de-N-acetylation of N-acetyl-d-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn2+ > Cu2+ > Ni2+ > Co2+ > Mg2+. Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.

Probing enzymes late in the trypanosomal glycosylphosphatidylinositol biosynthetic pathway with synthetic glycosylphosphatidylinositol analogues

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundant in the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness in humans and the related disease Nagana in cattle, and disruption of GPI biosynthesis is genetically and chemically validated as a drug target. Here, we examine the ability of enzymes of the trypanosomal GPI biosynthetic pathway to recognize and process a series of synthetic dimannosyl-glucosaminylphosphatidylinositol analogues containing systematic modifications on the mannose residues. The data reveal which portions of the natural substrate are important for recognition, explain why mannosylation occurs prior to inositol acylation in the trypanosomal pathway, and identify the first inhibitor of the third alpha-mannosyltransferase of the GPI biosynthetic pathway.

Specificities of Enzymes of Glycosylphosphatidylinositol Biosynthesis in Trypanosoma brucei and HeLa Cells

A series of synthetic analogues ofd-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol and the 4′-epimer,d-GalN-α1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the α-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the β-linked analogued-GlcNβ1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol α-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of thed-myo-inositol residue, whereas the human inositol acyltransferase and/or first α-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place ofsn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.

Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors.

1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system.

Further probing of the substrate specificities and inhibition of enzymes involved at an early stage of glycosylphosphatidylinositol (GPI) biosynthesis

1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1-O-hexadecyl-myo-inositol (14), 1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(octadecyl phosphate) (18), 1-D-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (24), 1-D-6-O-(2-amino-2-deoxy-alpha-D-mannopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (30) and the corresponding 2-amino-2-deoxy-alpha-D-galactopyranosyl analogue 36 have been prepared and tested in cell-free assays as substrate analogues/inhibitors of alpha-(1 --> 4)-D-mannosyltransferases that are active early on in the glycosylphosphatidylinositol (GPI) biosynthetic pathways of Trypanosoma brucei and HeLa (human) cells. The corresponding N-acetyl derivatives of these compounds were similarly tested as candidate substrate analogues/inhibitors of the N-deacetylases present in both systems. Following on from an early study, 1-L-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-methyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (44) was prepared and tested as an inhibitor of the trypanosomal alpha-(1 --> 4)-D-mannosyltransferase. A brief summary of the biological evaluation of the various analogues is provided.