Table 2.

Summary of commonly used strategies for assessing lysosomal GCase.

Assay	Substrate Examples	Measures	Best Applications	Application for therapeutic development	Disadvantages
Recombinant protein In vitro activity	4-MUG1, ResGlu2, BODIPY Glucosylceramide	GCase activity of recombinant protein	Analyzing direct effects of different environments/compounds on GCase enzyme kinetics	High throughput screening for GCase activators. Confirming lack of inhibitory activity for chaperones.	Does not account for variation in endogenous lysosomal factors that can affect activity
Cell Lysate in vitro activity	4-MUG, ResGlu BODIPY Glucosylceramide	Total GCase protein that includes lysosomal and non-lysosomal GCase	Analyzing total GCase protein, the effect of GCase mutations and covalent modification on GCase activity	Proof of concept studies for GCase chaperones and gene therapies	Is not able to correct for difference in GCase levels, which affect measured activity
Patient Biofluid in vitro activity	4-MUG, ResGlu	Total GCase protein	Activity measurement in serum and CSF	Evaluation of target engagement, Patient selection	Function of GCase in serum and CSF and correlation with tissue activity is unknown
Western Blotting	Antibody	Total GCase protein, ER GCase, post-ER GCase	Quantifying ER retention of GCase and post-ER GCase	Proof of concept studies for GCase chaperones and gene therapies	Does not report on enzyme activity
Inhibody	MDW333, MDW941	Lysosomal GCase protein	Quantifying lysosomal GCase protein, Analyzing GCase protein by microscopy	Proof of concept studies for GCase chaperones and gene therapies	Quantifies levels of active protein not enzyme activity
In situ GCase activity – cell culture	PFB-FDGlu3	In situ lysosomal GCase activity	Analyzing lysosomal GCase activity while accounting for endogenous factors	Screening, proof of concept studies for GCase chaperons, gene therapies and activators	Measurement will be affected by differences in substrate uptake
In situ GCase activity – PBMC	PFB-FDGlu	In situ lysosomal GCase activity	Analyzing lysosomal GCase activity while accounting for endogenous factors	Verify target engagement of chaperones and activators, patient selection	Measurement will be affected by differences in substrate uptake
Dry Blood Spot assay	C12 Glucosylceramide	Total GCase protein that includes lysosomal and non-lysosomal GCase	Analyzing total GCase protein, the effect of GCase mutations, covalent modification on GCase activity	Patient selection, target engagement of GCase chaperones	Requires specialized sample preparation and equipment. Does not account for variation in endogenous lysosomal factors

¹4-MU (4-methylumbelliferyl-β-D-glucopyranoside)

²ResGlu (Reresorufin-β-D-glucopyranoside)

³PFB-FDGlu (5-(Pentafluorobenzoylamino) Fluorescein Di-beta-D-Glucopyranoside)