Functionalized Cyclophellitols Are Selective Glucocerebrosidase Inhibitors and Induce a Bona Fide Neuropathic Gaucher Model in Zebrafish

Abstract

Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson's disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.

Figure 1

(a) Glucocerebrosidase (GBA) hydrolyses glucosylceramide in a two-step double displacement mechanism to yield glucose and ceramide. (b) Chemical structure of CBE 1 and cyclophellitol 2. (c) Mechanism-based inactivation of GBA by glucopyranoside-configured cyclitol epoxides (shown for cyclophellitol). (d) Structures of C8-extended cyclophellitol derivatives used in the here-presented studies: GBA activity-based probes ABPs 3–5 and selective inhibitors 6 and 7 (see the full chemical structures of ABPs 3–5 and 8–14 in the Supporting Information (SI)).

Figure 2

Structure of GBA reacted with ABP 5 and adamantyl-cyclophellitol 7. (a) GBA dimer, with the cyclophellitol and linker moiety of 5 shaded in yellow and a single observed Cy5 in pink. (b) Zoomed view of a GBA monomer reacted with ABP 5. (c) Structure of GBA with adamantyl-cyclophellitol 7. The linker-adamantyl moiety of 7 is observed in slightly different positions in the two molecules of the asymmetric unit (PDB 6Q6L, SI, Figure S2), reflecting its binding through predominantly hydrophobic interactions.

Figure 3

(a) Apparent IC₅₀ values for in vitro inhibition of GBA, GBA2, and GAA in recombinant enzymes (rGBA and rGAA) or overexpressed cell lysates (GBA2) by compounds 1, 2, 3, 5, 6, and 7. Error ranges depict standard deviations from biological triplicates. (b) Apparent IC₅₀ values for in vivo inhibition in 5-day treated zebrafish embryo with compounds 1, 2, 3, 5, 6, and 7. Error ranges depict standard deviations from n = 12–24 individuals. (c) Competitive ABPP in lysates of zebrafish treated in vivo with compounds 6 and 7 using broad-spectrum retaining β-glucosidase ABP 8 and selective GBA ABP 5 as readout. (d) Glucosylsphingosine levels produced in zebrafish embryos treated for 5 days with inhibitors 6, 7 or CBE 1. Error ranges depict standard deviations from n = 3 individuals. N/A, not analyzed; *, p < 0.5; ***, p < 0.001.

Figure 4

In vivo targets of ABP 3 and 7 in brains of adult zebrafish. Competitive ABPP in adult zebrafish homogenates with selective GBA ABP 5 (a) or broad-spectrum retaining β-glucosidase ABP 8 (b) as read-out.