Summary /

To demonstrate chaperone activity, the capacity of ML156 and ML155 to increase the translocation of GC to the lysosome was measured (8, 16, 30, 31). In this experiment, wildtype and mutant fibroblasts were incubated for five days with compound 8i, followed by cell fixation and staining with a selective fluorescent GC antibody. Compounds able to promote trafficking from the ER to the lysosome increased the fluorescent lysosomal signal. DMSO and isofagomine were used as negative and positive controls. Figure 2 shows the increment of signal in both cell lines that resulted from treatment, confirming the chaperone capacity of these compounds.

Figure 2: Chaperone activity of compound 13, NCGC00182292 (CID 40225210), and others using wildtype and homozygous, mutant N370S GC fibroblasts. Two genotypes of fibroblasts, fibroblasts homozygous for wildtype GC (top) and fibroblasts homozygous for N370S GC (bottom) were stained both with a Cy3-labeled antibody for GC protein content (first row) and a FITC-labeled antibody specific for lysosomal compartments (LAMP1; second row) after treatment with (1) DMSO vehicle, (2) 10μM Isofagomine (CID 447607), (3) 10μM compound 13, NCGC00182292 (CID 40225210), and (4) 1μM NCGC00159568 (CID 9893924). Several compounds, including isofagomine (CID 447607) and compound 13, NCGC00182292 (CID 40225210), show increased lysosomal GC protein after treatment.

Summary /

In enzyme kinetics assays, the probe, along with other glucocerebrosidase inhibitors such as isofagomine demonstrated mixed mode inhibition – neither competitive nor non-competitive enzyme kinetics. It is obvious, from x-ray crystal structure studies of isofagomine binding, that the compound binds in the active site. Thus, the current homogenate-based assays might be ill-suited for such kinetic characterization.