Azasugar inhibitors as pharmacological chaperones for Krabbe disease

Abstract

Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure-activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.

Fig. 1. Structure of GALC and azasugar compounds. (A) Ribbon diagram of GALC illustrating the β-sandwich (red), TIM-barrel (blue) and lectin (green) domains with β-d-galactose in the active site and N-linked glycans (pink sticks). Hydrogen bonding interactions (dashed lines) between galactose and active site residues are shown (zoomed box). (B) Pyranose ring numbering of β-d-galactose illustrating the anomeric position and chemical diagrams of the molecules used in this study: iso-galacto-fagomine (IGF); aza-galacto-fagomine (AGF); iso-galacto-fagomine lactam (IGL); dideoxyiminolyxitol (DIL); deoxy-galacto-noeurostegine (DGN) and deoxy-galacto-nojirimycin (DGJ). Compounds are color coded as indicated throughout this study.

Fig. 2. Synthesis of galacto-configured azasugars. (A) Synthesis of key intermediate 11 from 2,3-O-isopropylidene-d-ribofuranose (5). (B) Synthesis of IGF, IGL, DIL and AGF from a common intermediate (11).

Fig. 3. Competitive inhibition kinetics of galacto-configured azasugars. Plots of GALC initial velocity vs. substrate concentration with (A) DGJ, (B) IGF, (C) DIL, (D) AGF, (E) IGL and (F) DGN at concentrations encompassing the K _i (see ESI†). Inset plots of K _m^obs vs. concentration showing –K _i as the X-intercept. SEM error bars are shown.

Fig. 4. Stabilization of GALC by azasugar compounds. (A) Melt curves of GALC alone (black) and with 5.0 mM compound (chemical diagrams and coloring as above). Experiments were performed in sodium acetate, pH 4.6 and PBS, pH 7.4. (B) Bar graph showing the mean ΔT _m ± SEM conferred by 5.0 mM compound at pH 4.6 and pH 7.4. Experiments were performed in triplicate. (C) Values for mean ΔT _m ± SEM are tabulated. (D) Relationship between free energy of compound binding (ΔG) and GALC stabilization ΔT _m at pH 4.6. ΔG values for each compound were calculated from the measured K _i (Fig. 3) using the equation ΔG = RT ln K _i, where R is the gas constant (8.31 J K^–1 mol^–1) and T is absolute temperature (310 K).

Fig. 5. Structures of chaperone molecules bound in the GALC active site. X-ray crystal structures of (A) IGF, (B) IGL, (C) AGF and (D) DIL bound in the GALC active site. Compounds are colored as indicated. (Left panel) Unbiased difference electron density maps (F _O – F _C, 3.0σ, green) before ligand modelling at the active site of GALC. (Centre panel) Detail of the GALC active site with bound ligand, showing active site residues (sticks) and refined 2mF _O – DF _C electron density contoured at 0.25 e Å^–3 (blue). (Right panel) Schematic representation of hydrogen bond interactions (green dashed lines) between GALC and ligands.

Fig. 6. DGJ binds the active site in a distorted ¹ S ₃ conformation. (A) Unbiased difference electron density map (F _O – F _C, 3.0σ, green) before DGJ ligand refinement. (B) GALC active site with bound ligand, showing active site residues (sticks) and refined, feature-enhanced electron density map (0.65 e Å^–3, purple). (C) Schematic representation of hydrogen bond interactions (green lines) between GALC and DGJ. (D) Adoption of the ¹ S ₃ twisted-boat conformation allows DGJ to form a hydrogen bond with the nucleophile E258.

Fig. 7. DGN destabilizes GALC due to a steric clash in the active site. (A) Unbiased difference electron density map (F _O – F _C, 3.0σ, green) before DGN ligand refinement. (B) GALC active site with bound ligand, showing active site residues (sticks) and refined 2mF _O – DF _C electron density map (0.25 e Å^–3, blue). (C) Schematic representation of hydrogen bond interactions (green lines) between GALC and DGN. (D) Unbiased difference electron density (F _O – F _C; +3.0σ green, –3.0σ red) illustrating the steric clash between the ethylene bridge of DGN and W524 of the GALC lectin domain.

Fig. 8. Azasugar interaction with E258 is essential for optimal GALC stabilization. (A) Alignment of azasugar compounds bound in the GALC active site. Refined structures are overlaid, colored as indicated. Hydrogen bonds to catalytic residues E258 and E182 are shown (yellow lines). (B and C) Overlaid melt curves of GALC E258Q alone (black) and with 10 mM compound (colored as above). Experiments were performed in (B) sodium acetate, pH 4.6 and (C) PBS, pH 7.4.