salicylic hydroxamic acid as an inhibitor of Trypanosoma brucei GPI GlcNAc-PI de-N-acetylase

Abstract

The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions.

 

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Keywords

• GPI;
• Trypanosoma brucei;
• Hydroxamic acid;
• Inhibitor;
• N-Deacetylase

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1. Introduction

Trypanosoma brucei is a protozoan parasite that is transmitted by the bite of an infected tsetse fly and causes the fatal African sleeping sickness in humans and the related wasting disease Nagana in cattle.

 

Current treatments are expensive, toxic, and difficult to administer, leaving an urgent need for improved therapeutic agents.

 

The parasite is able to evade the host immune response by virtue of a dense surface coat composed of 5 × 10⁶ variant surface glycoprotein (VSG) dimers that prevent lysis by the innate immune response. The coat also undergoes antigenic variation to evade the adaptive immune response. ¹

 

The VSG dimer is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, and GPI anchor biosynthesis has been genetically and chemically validated as essential for the survival of the clinically relevant bloodstream form of the parasite. ^{2, 3, 4 and 5}

 

 

 

The GPI biosynthetic pathway has been extensively studied in both T. brucei and mammalian systems, highlighting differences in both the order of assembly and substrate specificity that can be exploited to produce species-specific inhibitors. The GlcNAc-PI de-N-acetylase is a zinc metalloenzyme that catalyses the second step in biosynthesis of GPI ( Fig. 1), ⁶

 

 

and has been genetically validated as essential in bloodstream form T. brucei. ³ The use of synthetic substrate analogues has revealed that the human enzyme is more fastidious than the trypanosome enzyme, enabling the design of species-specific substrate-based inhibitors. ^{7, 8 and 9} The substrate analogue approach has shown that the phosphate, 2′-NHAc and 3′-OH of the substrate GlcNAc-PI are critical for recognition by the T. brucei GlcNAc-PI de-N-acetylase. ¹⁰

 

 

In contrast, the diacylglycerol is not directly recognised and may be efficiently replaced with an octadecyl chain. ^9 and 11 Recently, we have shown that substrate-based inhibitors containing the zinc binding moieties hydroxamic acid or carboxylic acid can act as inhibitors of the T. brucei de-N-acetylase. ^12 and 13

 

 

These substrate-based inhibitors do not possess drug-like physiochemical properties and contain too much stereochemical complexity for tractable synthesis. Here, we report our efforts to identify alternative scaffolds for GlcNAc-PI de-N-acetylase inhibitors that resulted in the identification of salicylic hydroxamic acid as an inhibitor with high ligand efficiency.

 

 

 

 

 

Figure 1. 

GlcNAc-PI de-N-acetylase catalyses the second step in the GPI biosynthetic pathway. The zinc-dependent metalloenzyme is inhibited by zinc chelators.