A dual-functional substrate for quantitation of substrate levels and GCase activity in living cells

Loss of function mutations in the gene GBA1, which encodes the lysosomal glycoside hydrolase β-glucocerebrosidase (GCase) cause Gaucher's disease (GD). Moreover, one mutant allele of GBA1 is the most common genetic risk factor for the development of Parkinson's disease (PD). To gain a better understanding how these mutations drive development of PD and how GCase is regulated within cells, the field needs chemical reporters of GCase activity that can be used within living cells. Fluorogenic substrates are one method that can be used to quantify enzyme activities within cells yet existing substrates for GCase have limitations. In particular, the inability to monitor cellular uptake of substrate limits the ability to disentangle impairments in uptake of substrate from impairments in lysosomal GCase activity. Here we report on the preparation and biological characterisation of LysoRF-GBA - a new chemical tool which can be used to quantitatively measure both the cellular levels of intact substrate and lysosomal GCase activity within lysosomes. We demonstrate that, by using LysoRF-GBA, endogenous GCase activity can be measured within live neuroblastoma cells. The selectivity of this substrate for GCase, relative to other cellular enzymes, was validated by genetic and pharmacological perturbation of GCase. By using LysoRF-GBA and concomitantly monitoring levels of both cleaved product and intact substrate, we were able to measure GCase engagement with a known pharmacological chaperone and discriminate between pharmacological agents that affect GCase activity from those that impair endocytosis. Further, the ability to monitor intracellular levels of intact LysoRF-GBA also enabled us to measure its time dependent accumulation within cells, providing insight into when steady state levels of this substrate are reached. LysoRF-GBA therefore shows high potential to be exploited as a tool for the discovery of compounds that could beneficially modulate its activity for benefit in diseases including PD.