Potent and Selective Cell‐Active Iminosugar Inhibitors of Human α‐N‐Acetylgalactosaminidase (α‐NAGAL)

AbstractGlycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N‐acetyl‐α‐galactosaminidase (α‐NAGAL), the GH responsible for cleaving terminal α‐N‐acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non‐carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α‐NAGAL selective (∼200‐fold) guanidino‐containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image‐based method to measure levels of the Tn‐antigen, a cellular glycoprotein substrate of α‐NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α‐NAGAL within cells using patient derived fibroblasts (EC50=150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal β‐hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off‐target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α‐NAGAL.