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‐MUG, ResGlu, 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 nonlysosomal 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‐FDGlu | 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 nonlysosomal 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 |
Abbreviations: 4‐MUG, 4‐methylumbelliferyl‐β‐D‐glucopyranoside; ResGlu, Reresorufin‐β‐D‐glucopyranoside; BODIPY, boron dipyrromenthene; GCase, glucocerebrosidase; CSF, cerebrospinal fluid; PFB‐FDGlu, 5‐(Pentafluorobenzoylamino) Fluorescein Di‐β‐D‐Glucopyranoside; ER, endoplasmic reticulum; PBMC, peripheral blood mononuclear cell.