Mechanism-based inhibitors as in vitro and in vivo probes of glycosidase structure and mechanism

• Main Author: McCarter, John D.
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/7515

Two new classes of specific, mechanism-based glycosidase inactivators were developed: 2,2-dihalo glycosyl chlorides and 5-fluoro glycosyl fluorides. Both classes were effective against x-glucosidases, which had been hitherto resistant to similar inactivation strategies. Incubation of yeast -glucosidase with 2-chloro-2-deoxy-2-fluoroa- D-glucopyranosyl chloride or 2-deoxy-2,2-difluoro-α-D-arabinohexopyranosyl chloride resulted in time-dependent inactivation of the enzyme, presumably by formation of extremely stabilized 2,2-dihalo glycosyl-enzyme intermediates that are essentially incapable of turnover. Similar inhibition of Agrobacterium faecalis β-glucosidase and yeast α glucosidase was seen with the corresponding 5-fluoro glycosyl fluorides. 5-Fluoro-β- and 5-fluoro-α-D-glucosyl fluorides form catalytically competent intermediates with the appropriate glucosidases that are capable of turnover, but at rates reduced 10⁵ - and 10³ - fold, respectively, with respect to the β- and α-D-glucosyl fluoride parent substrates. The corresponding 5-fluoro-L-idosyl fluorides, the C5 epimers of the glucosyl compounds, show even greater reductions in turnover rates, the kcat values of the 5-fluoro-α- and 5- fluoro-β-L-idosyl fluorides with the appropriate enzymes being reduced a further 1.5- and 3000-fold. The spontaneous hydrolysis rates of these 5-fluoro glycosyl fluorides, and those of the corresponding 2-deoxy-2-fluoro compounds, were determined to probe the effects of the various fluorine substitutions on the transition states for hydrolysis. A novel mass spectrometric technique for the identification and sequencing of labelled active site peptides without the need for radiolabels has been developed. Briefly, the technique involves enzyme inactivation, proteolytic digestion of the enzyme, and identification of the modified peptide using electrospray tandem mass spectrometry by exploiting the lability of the inhibitor-peptide bond which is selectively cleaved by collisioninduced fragmentation. The key catalytic nucleophiles in the clinically important human lysosomal β-glucosidase (deficient in Gaucher disease), human acid β-galactosidase (deficient in GM1 gangliosidosis), and yeast α-glucosidase were identified, as Glu-340, Glu-268, and Asp-214, respectively. β-Glucosidase and β-mannosidase inhibitors, labelled with the positron-emitting isotope ¹⁸F, were synthesized for use in a novel approach to the in vivo imaging of glycosidase activity using positron emission tomography (PET). This may be useful in the diagnosis and treatment of abnormal glycosidase activity associated with disease (e.g. Gaucher disease, the inherited deficiency of lysosomal β-g1ucosidase). Initially, Agrobacterium faecalis β-glucosidase was labelled in vitro with an ¹⁸F-labelled mechanism-based enzyme inactivator 2-deoxy-2-[¹⁸F]fluoro--β-D-mannosyl[¹⁸F]fluoride, the first such labelling of a glycosidase with this positron-emitting isotope, and reactivation of the labelled enzyme was observed by monitoring release of radioactivity. Non-labelled versions of these inhibitors were administered to rats. Rapid inactivation of the appropriate enzymes was observed in each tissue assayed, and the clearance of the inhibitors was demonstrated by their slow release from the enzymes both in vitro and in vivo. Uptake and clearance of the inhibitors was probed using ¹⁹F NMR and ¹⁸F-radiolabelled compounds, revealing little hydrolysis but non-specific uptake of the inhibitor. Preliminary imaging results were obtained in rats using PET, demonstrating the potential of this approach for imaging glycosidase activity in vivo.