John S. Brimacombe

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.

Glycosyl-phosphatidylinositol molecules of the parasite and the host.

The glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10-20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.

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 synthesis of some seven-carbon sugars via the osmylation of olefinic sugars

Abstract The stereochemical outcome of the catalytic osmylation of 6,7-dideoxy-1,2:3,4-di- O -isopropylidene-α- d - galacto -hept-6-enopyranose ( 10 ), 5,6-dideoxy-1,2- O -isopropylidene-α- d - xylo -hex-5-enofuranose, ( E )- and ( Z )-3- O -benzyl-5,6-di-deoxy-1,2- O -isopropylidene-α- d - xylo -hept-5-enofuranose ( 20 and 27 , respectively), methyl ( Z )-3- O -benzyl-5,6-dideoxy-1,2- O -isopropylidene-α- d - xylo -hept-5-enofuranuronate ( 26 ), ( E )-3- O -benzyl-5,6-dideoxy-1,2- O -isopropylidene-α- d - ribo -hept-5-enofuranose, benzyl ( E )- and ( Z )-5,6-dideoxy-2,3- O -isopropylidene-α- d - lyxo -hept-5-enofuranoside ( 46 and 50 , respectively), and methyl [benzyl ( Z )-5,6-di-deoxy-2,3- O -isopropylidene-α- d - lyxo -hept-5-enofuranosid]uronate ( 49 ) has been examined. Such oxidations led to satisfactory syntheses of l - glycero - d - gluco -heptose and the corresponding heptitol (from 20 ), l - glycero - d - gulo -heptitol (from 26 ), d - glycero - d - gluco -heptitol (from 27 ), d - glycero - d - galacto -heptitol (from 10 and 46 ), ( meso )- glycero-gulo -heptitol (from 49 ), and d - glycero - d - manno -heptitol (from 50 ).

Nucleophilic displacement reactions in carbohydrates : part XII1. The reaction of 6-deoxy-2,3-O-isopropylidene-4-O-methanesulphonyl-α-L-talopyranose with sodium methoxide

Abstract Oxidation of benzyl 6-deoxy-2,3- O -isopropylidene-α-l-mannopyranoside (3) with ruthenium tetroxide in carbon tetrachloride gave benzyl 6-deoxy-2,3,- O -isopropylidene-α- l - lyxo -hexopyranosid-4-ulose ( 4 ) in excellent yield. Ketone 4 was reduced stereospecifically, with sodium borohydride in methanol, to yield benzyl 6-deoxy-2,3- O -isopropylidene-α- l -talopyranoside ( 5 ), which was converted into the crystalline 4-methanesulphonate 6 . Catalytic debenzylation of methanesulphonate 6 gave 6-deoxy-2,3- O -isopropylidene-4- O -methanesulphonyl-α- l -talopyranose ( 7 ), which, on solvolysis with sodium methoxide in methanol at room temperature, was converted into 1,4-anhydro-6-deoxy-2,3- O -isopropylidene-α- l -mannopyranose (1,5-anhydro-6-deoxy-2,3- O -isopropylidene-β- l -mannofuranose) ( 9 , 58%), methyl 6-deoxy-2,3- O -isopropylidene-α- l -talofuranoside ( 12 , 26%), and methyl 6-deoxy-2,3- O -isopropylidene-α- l -mannofuranoside ( 14 , 12%). The mechanisms of formation of these products are discussed.

Studies related to the synthesis of derivatives of 2,6-diamino-2,3,4,6-tetradeoxy-D-erythro-hexose (purpurosamine C), a component of gentamicin C12

Abstract Reduction of 1,6-anhydro-3,4-dideoxy-β- D - glycero -hex-3-enopyranos-2-ulose (levoglucosenone) with lithium aluminium hydride afforded principally 1,6-anhydro-3,4-dideoxy-β- D - threo -hex-3-enopyranose ( 3 ), which was converted into 3,4-dihydro-2( S )-hydroxymethyl-2 H -pyran ( 8 ) following acid-catalysed methanolysis and reductive rearrangement of the resulting α-glycoside 4 with lithium aluminium hydride. 1,6-Anhydro-3,4-dideoxy-2- O -toluene- p -sulphonyl-β- D - threo -hexopyranose, prepared from 3 , reacted slowly with sodium azide in hot dimethyl sulphoxide to give 1,6-anhydro-2-azido-2,3,4-trideoxy-β- D - erythro -hexopyranose, which was transformed into a mixture of methyl 2-acetamido-6- O -acetyl-2,3,4-trideoxy-α- D - erythro -hexopyranoside ( 10 ) and the corresponding β anomer following acid-catalysed methanolysis, catalytic reduction, and acetylation. Acid treatment of methyl 4,6- O -benzylidene-3-deoxy-α- D - erythro -hexopyranosid-2-ulose yielded the enone 15 , which was readily transformed into methyl 6- O -acetyl-3,4-dideoxy-α- D - glycero -hexopyranosid-2-ulose ( 19 ). Procedures for the conversions of DL - 8 , 10 , and 19 into methyl 2,6-diacetamido-2,3,4,6-tetradeoxy-α- D - erythro -hexopyranoside (methyl N,N′ -di-acetyl-α-purpurosaminide C) have already been described.

An approach to branched-chain amino sugars, particularly derivatives of l-vancosamine (3-amino-2,3,6-trideoxy-3-C-methyl-l-lyxo-hexose) and its d enantiomer, via the cyanohydrin route

Abstract Methyl 4,6- O -benzylidene-2-deoxy-α- d - erythro -hexopyranosid-3-ulose reacted with potassium cyanide under equilibrating conditions to give, initially, methyl 4,6- O -benzylidene-3- C -cyano-2-deoxy-α- d - ribo -hexopyranoside ( 7 ), which, because it reverted slowly to the thermodynamically stable d - arabino isomer, could be crystallised directly from the reaction mixture. The mesylate derived from the kinetic product 7 could be converted by published procedures into methyl 3-acetamido-2,3,6-trideoxy-3- C -methyl-α- d - arabino -hexopyranoside, which was transformed into methyl N -acetyl-α- d -vancosaminide on inversion of the configuration at C-4. A related approach employing methyl 2,6-dideoxy-4- O -methoxymethyl-α- l - erythro -hexopyranosid-3-ulose gave the kinetic cyanohydrin and thence, via the spiro-aziridine 27 , methyl 3-acetamido-2,3,6-trideoxy-3- C -methyl-α- l - arabino -hexopyranoside, a known precursor of methyl N -acetyl-α- l -vancosaminide.