Enzymes mediate a great diversity of reactions that enable a wide range of cellular processes. The activities of enzymes within cells often vary depending upon the tissue type and developmental stage. Many enzyme-mediated processes within tissues are coordinated through a range of mechanisms that regulate their activities [1]. Unsurprisingly, the dysregulation of enzymes often drives disease. Therefore, studying enzymes within their native cellular environments, where they are subject to these regulatory mechanisms, is important. However, the vast majority of enzymatic studies rely on in vitro experiments using recombinant proteins or cell lysates. Although informative, these approaches cannot fully capture the complex native environments found within cells, nor can they readily report on the cellular processes involved in their regulation. As such, there is a need for new assay modalities that can be used to measure enzyme activities within their native milieus.

Recognising the potential of this area, chemical biologists have created new synthetic enzyme substrates that undergo photophysical changes upon their processing by various enzymes including, for example, proteases [2], thioesterases [3], reductases [4], esterases [4], phosphatases [4], and glycoside hydrolases (GHs) [4]. The consequent changes in the photophysical properties of these substrates can be observed using various imaging techniques, often directly within cells or tissues of interest. While the contents of this review can be generally applied, we will focus here on cell-active substrates for members of the superfamily of GHs, with an emphasis on the set of mammalian enzymes.

Glycoside hydrolases are a superfamily of enzymes that have been conveniently categorised, based on sequence similarities, into over 150 families that share common activities and enzyme active site architectures [5]. Within humans, there are 87 GH encoding genes, of which 30 have so far been implicated in congenital human diseases (Figure 1) [6]. Many of these congenital diseases are lysosomal storage diseases wherein loss of function mutations cause the build-up of glycoconjugate substrates within the lysosomes of certain cell types [7]. However, as GHs play fundamental roles in many other biological processes such as protein folding, cell signaling, and energy metabolism, modulating the activities of these enzymes can influence various idiopathic and polygenic diseases. For example, antagonists of the GH84 enzyme O-GlcNAcase have been found to hinder progression of various neurodegenerative diseases [8] and compounds inhibiting the GH116 non-lysosomal glucosylceramidases (GBA2) are therapeutics for Niemann-Pick type C disease [9]. Most recently, loss of function mutations in one allele of genes encoding certain lysosomal GHs have been genetically linked to the synucleinopathies [10]. Despite growing recognition of the GHs as disease relevant enzymes, the mechanisms by which many of these enzymes contribute to their associated pathologies remain poorly understood.

How GHs are regulated within cells is an underexplored area, but for reasons noted above, is of increasing fundamental interest. Notable for GHs is the recurring importance of regulatory partners, which are in many cases themselves disease-associated or disease modifying genes. In addition to these partners, the potential for control of GHs within cells by post-translational modifications is an intriguing area [11]. Accordingly, a better understanding of GHs activities and regulation requires the ability to study them within their biologically relevant contexts. Furthermore, one common approach to treating mutations that compromise the folding and trafficking of these enzymes is to use pharmacological chaperones [12], several of which have reached the clinic and in one case gaining approval and seeing benefits to patients [13]. These pharmacological chaperones, however, are typically enzyme inhibitors and so being able to monitor inhibition in situ within cells would be a great aid in their development. Accordingly, new cell-compatible tools and methodologies should provide new fundamental insights and, ultimately, aid the development of new therapeutics. In this review we focus on the most recent advances within the field, placing these in the context of key literature.