Abstract
A series of synthetic analogues of 1-D-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the D-myo-inositol, D-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The D-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the β-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.
Highlights
We showed that T. brucei GlcNAc-PI de-N-acetylase is a zinc-dependent metalloenzyme10 and demonstrated, by construction of a condition-null mutant cell line, that it is essential for the bloodstream form of the parasite and, a genetically validated drug target
The separation of anomers was achievable at this stage and these anomers were vital in providing the glucosamine-phosphodiester target analogues discussed
For the sake of brevity, we have chosen to describe in the main text the formation of the α-anomers, while details for the corresponding β-anomers appears in the Electronic supplementary information (ESI).† the pseudodisaccharide 16 was coupled to the hydrogen phosphonate 18,15 and the ensuing mixture of diastereoisomeric
Summary
The enzymes of the glycosylphosphatidylinositol (GPI) biosynthetic pathway are located in the endoplasmic reticulum, contain between one and thirteen predicted trans-membrane domains and are mostly present as components of multisubunit complexes.1 No high-resolution structural data are available on any of these enzymes and our research group has been probing the specificities of several of the enzymes in the GPI pathway of the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness in humans and the related disease Nagana in cattle, using synthetic substrate analogues in vitro.2–8 One of the key enzymes of interest is an amidase, the GlcNAc-PI de-N-acetylase (EC 3.5.1.89) that deN-acetylates 1-D-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (1, α-D-GlcpNAc-PI) to 1-D-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (2, α-D-GlcpN-PI), Fig. 1.This enzyme catalyses the second step in the T. brucei GPI biosynthetic pathway, which is a prerequisite for all subsequent steps in the pathway.9 In earlier studies, we showed that T. brucei GlcNAc-PI de-N-acetylase is a zinc-dependent metalloenzyme10 and demonstrated, by construction of a condition-null mutant cell line, that it is essential for the bloodstream form of the parasite and, a genetically validated drug target.11 Previous studies with other substrate analogues showed that the phosphate, 2′-NHAc and 3′-OH groups of the natural substrate α-D-GlcpNAc-PI [1] are critical for recognition by the T. brucei GlcNAc-PI de-N-acetylase.2–4 In contrast, the diacylglycerol moiety is not strictly required and may be efficiently replaced with an octadecyl chain,4 as shown in analogues 3 and 4.
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