Figure 1.
pH profiles of recombinant N370S and WT GCase.
The assay was performed in the presence of 10 mM NaTC. While the WT enzyme has a pH optimum of 5.9, the N370S enzyme has a broad optimum of around pH 7.2.
Figure 2.
Concentration responses of three GCase inhibitors determined using recombinant WT GCase, recombinant N370S mutant enzyme, WT GCase from normal spleen extract and N370S mutant enzyme from a spleen of a patient with GD (genotype N370S/N370S).
The three inhibitors were identified from the previous HTS using recombinant WT GCase.
Figure 3.
Characterization of spleen GCase activity.
A. pH optimum, B. NaTC dependency, C. Inhibition of spleen GCase activity by CBE.
Figure 4.
Effect of adding the spleen preparation on the activity of WT recombinant GCase.
pH dependency and effect of sodium NaTC on the recombinant enzyme activity were assayed. A. activity of WT recombinant enzyme with and without NaTC. B and C. Different dilutions of N370S spleen in the presence (B) or absence (C) of NaTC. D. and E. WT recombinant enzyme mixed with increasing concentrations of spleen extract with (D) and without (E) NaTC.
Figure 5.
Effect of SapC versus NaTC on the activity of WT (A) and N370S recombinant (B) GCase.
In the presence of SapC, the activity of both WT (A), N370S (B) enzyme gave a sharp peak near pH 5, compared to the broader peak and higher pH seen using NaTC.
Table 1.
Classification of active compounds identified from the screen assay using the N370S mutant enzyme in spleen tissue.
Table 2.
Compounds discovered in the spleen qHTS tested for modulation of GCase activity and GCase translocation in cell assays.
Figure 6.
Dose response increase in lysosomal content of GCase in fibroblasts treated with selected identified compounds.
Cells were treated with six drug concentrations (1 nM to 50 µM) for 5 days. The intensity of GCase staining in the lysosomes was measured using an automatic fluorescent microscope. The slope of dose dependent increase in fluorescence was plotted and statistically significant slopes are labeled with (*). Some compounds, including the activator NCGC00182510, were able to increase the lysosomal content of both N370S and L444P mutant enzymes. Some compounds were found to be toxic at the highest concentration and that point was removed from calculations.
Figure 7.
Comparison of the active compounds identified in the WT and N370S HTS experiments.
A. Distribution of active compounds in the WT (blue) and N370S/N370S spleen (red) HTS experiments. B. Overlap between active compounds tested in both experiments by type of activity.
Figure 8.
Potency of selected compounds in the WT and N370S mutant enzyme assay with NaTC or SapC.
Compound NCGC0092410 (NCGC), discovered in the recombinant WT HTS, inhibited the WT enzyme much better than N370S in NaTC (A), but showed less inhibition of WT enzyme in the SapC assay (B). Compound MLS000393962 (MLS) (C,D), identified in the N370S/N370S spleen HTS, inhibited the WT recombinant enzyme, and had no effect on the recombinant N370S enzyme with the addition of NaTC (C). However, in the presence of SapC (D), there was an enhanced inhibitory effect on the WT enzyme as well as the N370S enzyme. * No trendline fit possible.