Abstract
Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β‐glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA‐targeting activity‐based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism‐based mode of action and uncover binding of the new N‐functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6‐fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6‐ and N‐functionalised reagents, a bi‐functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.
Highlights
Gaucher disease (GD) is the most common lysosomal storage disorder which is caused by inherited deficiencies in βglucocerebrosidase
A co-crystal structure of recombinant human GBA in complex with azide-tagged cyclophellitol 1 was obtained at 1.70 Å resolution, revealing classical trans-diaxial ring-opening of the epoxide warhead to form a covalent enzyme-inhibitor complex with the enzymatic nucleophile (Glu340), (Figure 2a)
An absence of azide electron density for inhibitor 1 has been reported previously when in complex with an unrelated bacterial β-glucoside (Thermotoga maritima, TmGH1).[54]. In contrast to this bacterial co-complex, the improved azido-electron density of 1 in complex with recombinant human GBA (rhGBA) reported here provides some insight into the conformation of the azide-tag, which may serve as a ligation handle for two step activity-based protein profiling (ABPP).[55,56]
Summary
Gaucher disease (GD) is the most common lysosomal storage disorder which is caused by inherited deficiencies in βglucocerebrosidase (glucosylceramidase, GCase, GBA, EC 3.2.1.45) This lysosomal glycoside hydrolase is encoded by the GBA1 gene[1] and according to The Human Gene Mutation Database (www.hgmd.org, Institute of Medical Genetics in Cardiff[2]) over 500 genetic mutations at the GBA1 locus are known. The enzymatic nucleophile of GBA was identified (and corrected from Asp443[19]) by the Wither’s lab through covalent-trapping of the enzyme with a 2-fluoro-2-deoxy glucoside inactivator.[20] Given the clinical importance of GBA in both GD and PD, it is arguably the most widely studied human glucosidase, with relentless interest in developing novel chaperones,[21,22,23,24] inhibitors[25,26,27] and activity-based probes (ABPs)[28,29] to study this enzyme in disease pathogenesis, diagnosis and treatment.
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