(Z)-Pugnac

(Z)-Pugnac is a potent inhibitor of O-GlcNAcase [1].

Pugnac is an inhibitor of O-GlcNAcase and is initially found to be an inhibitor of β-N-acetylglucosaminidases. It plays its inhibition role through mimicking the substrates of its target enzymes.(Z)-Pugnac is the Z stereoisomer of Pugnac. It is found to be more potent than the E stereoisomer. When tested with the recombinant O-GlcNAcase and the fluorogenic substrate, (Z)-Pugnac inhibited approximately 80% of O-GlcNAcase native activity at concentration of 1 μM. (E)-Pugnac at the same concentration showed only 10% inhibition of the activity. Besides that, (Z)-Pugnac induced about 1.4-fold increased level of O-GlcNAc incorporation on proteins both in HEK cells and in HeLa cells [1].

References:
[1] Perreira M, Kim E J, Thomas C J, et al. Inhibition ofO-GlcNAcase by PUGNAc is dependent upon the oxime stereochemistry. Bioorganic & medicinal chemistry, 2006, 14(3): 837-846.

Inhibition of O-GlcNAcase by PUGNAc is dependent upon the oxime stereochemistry.[Pubmed:16214344]

Bioorg Med Chem. 2006 Feb 1;14(3):837-46.

The potent O-GlcNAcase inhibitor PUGNAc was synthesized and two isomers based on the E and Z stereochemistry of the oxime moiety were separated, defined, and tested for activity. Several lines of evidence were examined in an effort to define the correct stereochemical assignments of each form of PUGNAc. The ability of the Z stereoisomer to undergo the Beckmann rearrangement was ultimately the most definitive proof. It was determined via both in vitro and intact cell experiments that the Z form of PUGNAc was vastly more potent an inhibitor of O-GlcNAcase than the E form.

O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors.[Pubmed:15795231]

J Biol Chem. 2005 Jul 8;280(27):25313-22.

The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of beta-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 beta-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal beta-hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl-beta-D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Consistent with this proposal, 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline, an inhibitor that mimics the oxazoline intermediate proposed in the catalytic mechanism of family 20 glycoside hydrolases, is shown to act as a potent competitive inhibitor of both O-GlcNAcase (K(I) = 0.070 microm) and beta-hexosaminidase (K = 0.070 microm). A series of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K(I) = 0.230 microm) over beta-hexosaminidase (K(I) = 340 microm). These inhibitors are cell permeable and modulate the activity of O-GlcNAcase in tissue culture. Because both enzymes have vital roles in organismal health, these potent and selective inhibitors of O-GlcNAcase should prove useful in studying the role of this enzyme at the organismal level without generating a complex chemical phenotype stemming from concomitant inhibition of beta-hexosaminidase.

Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol.[Pubmed:8034696]

J Biol Chem. 1994 Jul 29;269(30):19321-30.

Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient post-translational modification. To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-beta-D-glucosaminidase (O-GlcNAcase) with strong selectivity for O-GlcNAc-synthetic glycopeptides has been purified over 22,000-fold from rat spleen homogenate. The purified O-GlcNAcase has two major polypeptides of apparent M(r) = 54,000 (alpha subunit) and M(r) = 51,000 (beta subunit). Enzyme activity sediments at M(r) = 106,000 on sucrose gradients, indicating that the native O-GlcNAcase is an alpha beta heterodimer. The O-GlcNAcase also shows substantially stronger relative activity against O-GlcNAc-synthetic glycopeptides than other hexosaminidases. Unlike acidic lysosomal hexosaminidases, O-GlcNAcase is not inhibited by GalNAc or its analogs, has no other detectable glycosidase activities, and does not cross-react with antibodies against acidic hexosaminidases. Subcellular fractionation and latency studies demonstrate the cytoplasmic and nucleoplasmic localization of the enzyme and its ubiquitous presence in tissues. These studies suggest that O-GlcNAcase is involved in the regulated removal of O-GlcNAc from O-GlcNAc-bearing glycoproteins in the nucleoplasmic and cytoplasmic compartments of cells.