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

Glycoside 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-& alpha;-galactosaminidase (& alpha;-NAGAL), the GH responsible for cleaving terminal & alpha;-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 & alpha;-NAGAL selective (& SIM;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 & alpha;-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of & alpha;-NAGAL within cells using patient derived fibroblasts (EC50=150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal & beta;-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 & alpha;-NAGAL.