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

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in various diseases. 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 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 a-NAGAL. Using this assay we show that DGJGuan exhibits excellent inhibition of a-NAGAL within cells using patient derived fibroblasts (EC50 = 150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal b-hexosaminidase substrate ganglioside GM2 show that DGJGuan 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.