(A) Irreversible inhibition by cyclophellitol and cyclophellitol‐aziridine compounds. (B) Reactivity of GBA, GBA2 and GBA3 β‐d‐glucosidases with epoxide and aziridine‐based ABPs 9 and 10, R=Cy5.

Structures of cyclophellitol epoxide and aziridines subject of the research described in this paper.

Selectivity of compounds visualized by competitive ABPP labeling of β‐d‐glucosidase. Lysates of HEK293T cells expressing human GBA, GBA2 and GBA3 were incubated with compounds 1–5 at indicated concentrations for 30 min, following by cABPP with ABP 10.

(A) ABP labeling of β‐d‐glucosidases. A lysate of HEK293T cells expressing human GBA, GBA2 and GBA3 was incubated with indicated ABPs (6, 9 or 10) for 30 min at pH 6.0. Fluorescently labeled proteins were visualized after SDS‐PAGE. (B) Labeling with ABP 6 of wild type or mutant GBA2 (E527G nucleophile mutation and D667G acid/base mutation) expressed in HEK293T cells.

Inhibitory effect of β‐d‐xylo‐configured cyclophellitol 1 and cyclophellitol aziridine 2 on β‐glucosidases in intact HEK293T cells expressing GBA, GBA2 and GBA3. (A) Representative gel images of cABPP where cells were treated for 24 h with indicated inhibitor. Lysates were then prepared and labeled with fluorescent ABP 10. Fluorescently labeled proteins were visualized after SDS‐PAGE (1 set from n=3 replicates). (B) IC₅₀ curves determined by cABPP labeling results. (C) Apparent IC₅₀ values towards β‐glucosidases in intact HEK293T cells producing GBA, GBA2 and GBA3 were determined by the fluorescence quantification based on cABPP SDS‐PAGE results.

In vivo inhibitory effect of β‐d‐xylo‐configured compounds on β‐glucosidases in zebrafish (Danio rerio) larvae. (A) Larvae were exposed to 5 dpf with the indicated inhibitor 1 or 2 in the medium. Larvae were lysed and incubated with fluorescent ABP 10. Fluorescently labeled proteins were visualized after SDS‐PAGE, only GBA and GBA2 were assessed in zebrafish larvae model. (B) Apparent IC₅₀ values towards β‐glucosidases (GBA and GBA2) were determined by the fluorescence quantification based on cABPP results. (C) In vivo inhibition curves. (D) GlcSph levels in zebrafish larvae were determined as described in experiment section, n=2 replicates.