ABSTRACT
The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.
Subject(s)
Catalytic Domain , Computer Simulation , Hexosaminidase A/metabolism , Sugars/metabolism , Sugars/pharmacology , Tay-Sachs Disease/drug therapy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Hexosaminidase A/antagonists & inhibitors , Hexosaminidase A/chemistry , Hexosaminidase A/genetics , Humans , Molecular Dynamics Simulation , Mutation , Sugars/chemistry , Sugars/therapeutic useABSTRACT
Protein O-GlcNAc glycosylation is a ubiquitous post-translational modification in metazoans. O-GlcNAcase (OGA), which is responsible for removing O-GlcNAc from serine or threonine residues, plays a key role in O-GlcNAc metabolism. Potent and selective O-GlcNAcase (OGA) inhibitors are useful tools for investigating the role of this modification in a broad range of cellular processes, and may also serve as drug candidates for treatment of neurodegenerative diseases. Biological screening of the gluco-configured tetrahydroimidazopyridine derivatives identified a compound as a potent and competitive inhibitor of human O-GlcNAcase (OGA) with a K(i) of 5.9 µM, and it also displayed 28-fold selectivity for human OGA over human lysosomal ß-hexosaminidase A (Hex A, K(i)=163 µM). In addition, cell-based assay revealed that this compound was cell-permeant and effectively induced cellular hyper-O-GlcNAcylation at 10 µM concentration.
Subject(s)
Enzyme Inhibitors/chemistry , Imidazoles/chemistry , Pyridines/chemistry , beta-N-Acetylhexosaminidases/antagonists & inhibitors , Cell Line, Tumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glycosylation/drug effects , Hexosaminidase A/antagonists & inhibitors , Hexosaminidase A/metabolism , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Kinetics , Lysosomes/enzymology , Pyridines/chemical synthesis , Pyridines/pharmacology , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Gal-PUGNAc (see picture), a highly selective inhibitor for beta-hexosaminidases HEXA and HEXB is cell-permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal-PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O-GlcNAcase.
