3,4,5‐Trihydroxypiperidine Based Multivalent Glucocerebrosidase (GCase) Enhancers

Abstract The synthesis of five new multivalent derivatives of a trihydroxypiperidine iminosugar was accomplished through copper catalyzed alkyne‐azide cycloaddition (CuAAC) reaction of an azido ending piperidine and several propargylated scaffolds. The resulting multivalent architectures were assayed as inhibitors of lysosomal GCase, the defective enzyme in Gaucher disease. The multivalent compounds resulted in much more potent inhibitors than a parent monovalent reference compound, thus showing a good multivalent effect. Biological investigation of these compounds as pharmacological chaperones revealed that the trivalent derivative (12) gives a 2‐fold recovery of the GCase activity on Gaucher patient fibroblasts bearing the L444P/L444P mutations responsible for neuropathies. Additionally, a thermal denaturation experiment showed its ability to impart stability to the recombinant enzyme used in therapy.


S12
Hexavalent 14: Application of the general procedure for CuAAC reaction (reported in the main text) to scaffold 9 (1 equiv.) and 6.6 equiv. of 5 afforded 55% of 14 as a white waxy solid (  Hexavalent 15: Application of the general procedure for CuAAC reaction (reported in the main text) to scaffold 10 (1 equiv.) and 6.6 equiv. of 5 afforded 85% of 15 as a white waxy solid (

Synthesis of compound 18:
To a solution of 17 (73 mg, 0.18 mmol) in 8 mL of methanol, 150 µL of 37% HCl were added and the mixture was stirred at room temperature for 18 hours. After that a TLC analysis (CH2Cl2:MeOH 10:1) showed disappearance of the starting material (Rf = 0.59), the solvent was removed under reduced pressure. The product, obtained as its hydrochloric salt, was then treated with the strongly basic resin Ambersep 900-OH to afford the final product 18 (62 mg, 0.18 mmol, 95% yield) as a colourless oil.
Figure S19: Activity of GCase in the presence of compounds (1 mM). The corresponding calculated percentage of inhibition is indicated above each bar.

IC50 determination:
The IC50 values of inhibitors against GCase were determined by measuring the initial hydrolysis rate with 4-methylumbelliferyl-β-D-glucoside (3.33 mM). Data are mean ± SD (n=3). Data obtained were fitted to the following equation using the Origin Microcal program. where Vi/Vo, represent the ratio between the activity measured in the presence of the inhibitor (Vi) and the activity of the control without the inhibitor (V0), "x" the inhibitor concentration, Max and Min, the maximal and minimal enzymatic activity observed, respectively.

IC50 determination of 12 towards recombinant wild-type human GCase:
The IC50 value of 12 against recombinant wild-type human GCase enzyme (VPRIV®, enzyme was diluted in bovine serum albumin (0.2%) at final concentration of 1.0 x 10 -9 mg/mL) were determined by measuring the initial hydrolysis rate with 4-methylumbelliferyl-β-D-glucoside at different concentrations of 12.
A proper solution of 12 (3 μL), recombinant wild-type human GCase enzyme solution (7 μL) and substrate 4methylumbelliferyl-β-D-glucoside (3.33 mM, 20 μL, Sigma-Aldrich) in citrate/phosphate buffer (0.1:0.2, M/M, pH 5.8) containing sodium taurocholate (0.3%) and Triton X-100 (0.15%) at 37 °C were incubated for 1 h. The reaction was stopped by addition of sodium carbonate (200 μL; 0.5M, pH 10.7) containing Triton X-100 (0.0025 %), and the fluorescence of 4-methylumbelliferone released by β-glucosidase activity was measured in SpectraMax M2 microplate reader (λex=365 nm, λem=435 nm; Molecular Devices). Percentage GCase inhibition is given with respect to the control (without iminosugar). Data are mean ± SD (n=3). Data obtained were fitted accordingly to the above-mentioned equation and using the Origin Microcal program.  The mechanism of action of compound 12 was determined by studying the dependence of the main kinetic parameters (Km and Vmax) on the increase in inhibitor concentration. We observed that the Km value increased with increasing inhibitor concentration, while the Vmax did not change ( Figure S28). Furthermore, the double reciprocal plot showed that the experimental points described straight lines intersecting each other at a point on the ordinate axis ( Figure S27). Together, these data suggested that compound 12 behaves as a pure competitive inhibitor with respect to GCase. Therefore, using the appropriate equation, we calculated the value of the inhibition constant (Ki) which was 3.1 ± 0.2 µM ( Figure S29).

Chaperoning activity assays
Evaluation of the effect of multimeric iminosugars (12, 13,15) on GCase activity in Gaucher patients' cells: Fibroblasts with the N370S/RecNcil (or L444P/L444P) mutation from Gaucher disease patients were obtained from the "Cell line and DNA Biobank from patients affected by Genetic Diseases" (Gaslini Hospital, Genova, Italy). Fibroblasts cells (20 x10 4 ) were seeded in T25 flasks with DMEM supplemented with fetal bovine serum (10 %), penicillin/streptomycin (1%), and glutamine (1%) and incubated at 37 °C with 5% CO2 for 24 h. The medium was removed, and fresh medium containing the multimeric iminosugars was added to the cells and left for 4 days. The medium was removed, and the cells were washed with PBS and detached with tripsin to obtain cell pellets, which were washed four times with PBS, frozen and lysed by sonication in water. Enzyme activity was measured as reported above. Reported data are mean ± S.D. (n=2).