Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity

Abstract

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher's disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson's disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging.

Stabilization of Glucocerebrosidase by Active Site Occupancy

“…33,34 Along the same line, Vocadlo and co-workers developed an elegant fluorescence-quenched substrate for GBA by exploiting the fact that GBA accommodates the hydrophobic ceramide moiety of glucosylceramide, as it is known that GBA could tolerate a hydrophobic modification in the 6-position of glucose. 49 The designed high-affinity fluorescence-quenched substrate harbors a fluorophore attached at C6 of glucose and the hydrophobic quencher attached to the anomeric site; to a certain extent, it mimics fluoro 3 and ABPs 4 and 5 . This successful substrate design indicates again that the catalytic pocket of GBA accommodates substrates possessing a hydrophobic modification.…”

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease

“…Yadav and co-workers recently proposed a GCase-specific fluorescence-quenched substrate that favors lysosomal uptake with a quencher group attached to the long aliphatic chain of the ceramide moiety of GlcCer and a fluorophore group on the glucose part. The outcome of the close proximity of the quencher-fluorophore pair is efficient quenching (85). A live cell confocal fluorescence microscopy assay on wild type fibroblasts revealed a time-dependent increase in fluorescence signal in lysosomes due to substrate turnover and subsequent loss of quenching.…”

“…Unfortunately, treatment of fibroblasts derived from GD patients was missing from this study. Additionally, laser scanning confocal microscopy assays are not feasible for HTS purposes and more suitable read-out platforms such as a fluorescence plate reader platform were not included in this study (85). …”

Recent advances in the development of synthetic chemical probes for glycosidase enzymes

“…11). 113 Substrate 28 showed localisation within the lysosome and allowed the timedependent monitoring of endogenous GCase activity within human fibroblasts. Substrate 28 contains the quencher group Black Hole Quencher 2 (BHQ2) attached to the anomeric center using a spacer and the fluorophore BODIPY attached to the 6-position as modification at this position has been reported to be tolerated.…”