Potent O-GlcNAcase Inhibitor Research Articles

Structure-based discovery and development of novel O-GlcNAcase inhibitors for the treatment of Alzheimer's disease

The neurofibrillary tangles (NFTs) formed from hyperphosphorylation of tau protein are closely associated with Alzheimer's disease (AD). O-GlcNAcylation of tau can negatively regulate hyperphosphorylation and the O-GlcNAcase (OGA) catalyzes the removal of O-linked β-N-acetylglucosamine (O-GlcNAc) from tau protein. Therefore, preventing tau hyperphosphorylation by increasing the levels of tau O-GlcNAcylation via OGA inhibitors could be a promising approach. Based on Thiamet-G, a potent OGA inhibitor, and its binding mode to OGA, a novel OGA inhibitor scaffold bearing three parts was designed and hit compound 7j was successfully identified via extensive exploring. Further chemical optimization and diversification of the 7j structure resulted in compound 39 which possesses excellent OGA inhibition, no cytotoxicity, and has good pharmacokinetic properties. In acute AD model mice, 39 was more effective than Thiamet-G in inhibiting OGA activity attributable to its better blood-brain barrier permeability. In addition, 39 restored the cognitive function in mice and reduced amyloid-β (Aβ) concentrations to a greater extent than Thiamet-G. Molecular docking studies demonstrated that 39 was well associated with OGA through H-bonds and hydrophobic interaction. Together, these findings suggest that 39 was promising as a potent OGA inhibitor in the treatment of AD.

Discovery of MK-8719, a Potent O-GlcNAcase Inhibitor as a Potential Treatment for Tauopathies.

Inhibition of O-GlcNAcase (OGA) has emerged as a promising therapeutic approach to treat tau pathology in neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Beginning with carbohydrate-based lead molecules, we pursued an optimization strategy of reducing polar surface area to align the desired drug-like properties of potency, selectivity, high central nervous system (CNS) exposure, metabolic stability, favorable pharmacokinetics, and robust in vivo pharmacodynamic response. Herein, we describe the medicinal chemistry and pharmacological studies that led to the identification of (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diol 42 (MK-8719), a highly potent and selective OGA inhibitor with excellent CNS penetration that has been advanced to first-in-human phase I clinical trials.

Evaluation of a PET Radioligand to Image O-GlcNAcase in Brain and Periphery of Rhesus Monkey and Knock-Out Mouse.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.

Design and synthesis of O-GlcNAcase inhibitors via ‘click chemistry’ and biological evaluations

Protein O-GlcNAcylation has been shown to play an important role in a number of biological processes, including regulation of the cell cycle, DNA transcription and translation, signal transduction, and protein degradation. O-GlcNAcase (OGA) is responsible for the removal of O-linked β- N-acetylglucosamine (O-GlcNAc) from serine or threonine residues, and thus plays a key role in O-GlcNAc metabolism. Potent OGA inhibitors are useful tools for studying the cellular processes of O-GlcNAc, and may be developed as drugs for the treatment neurodegenerative diseases. In this study, Cu(I)-catalyzed ‘Click’ cycloaddition reactions between glycosyl azides and alkynes were exploited to generate inhibitory candidates of OGA. Enzymatic kinetic screening revealed that compound 7 was a potent competitive inhibitor of human O-GlcNAcase ( K i = 185.6 μM). Molecular docking simulations of compound 7 into CpOGA ( Clostridium perfringens OGA) suggested that strong π–π stacking interaction between the compound and W490 considerably contributed to improving the inhibitory activity.

Glucosamine-induced increase in Akt phosphorylation corresponds to increased endoplasmic reticulum stress in astroglial cells

Increased glucose flux through the hexosamine biosynthetic pathway (HBP) is known to affect the activity of a number of signal transduction pathways and lead to insulin resistance. Although widely studied in insulin responsive tissues, the effect of increased HBP activity on largely insulin unresponsive tissues, such as the brain, remains relatively unknown. Herein, we investigate the effects of increased HBP flux on Akt activation in a human astroglial cells line using glucosamine, a compound commonly used to mimic hyperglycemic conditions by increasing HBP flux. Cellular treatment with 8 mM glucosamine resulted in a 96.8% +/- 24.6 increase in Akt phosphorylation after 5 h of treatment that remained elevated throughout the 9-h time course. Glucosamine treatment also resulted in modest increases in global levels of the O-GlcNAc protein modification. Increasing O-GlcNAc levels using the O-GlcNAcase inhibitor streptozotocin (STZ) also increased Akt phosphorylation by 96.8% +/- 11.0 after only 3 h although for a shorter duration than glucosamine; however, the more potent O-GlcNAcase inhibitors O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline (NAGBT) failed to mimic the increases in phospho-Akt indicating that the Akt phosphorylation is not a result of increased O-GlcNAc protein modification. Further analysis indicated that this increased phosphorylation was also not due to increased osmotic stress and was not attenuated by N-acetylcysteine eliminating the potential role of oxidative stress in the observed phospho-Akt increases. Glucosamine treatment, but not STZ treatment, did correlate with a large increase in the expression of the endoplasmic reticulum (ER) stress marker GRP 78. Altogether, these results indicate that increased HBP flux in human astroglial cells results in a rapid, short-term phosphorylation of Akt that is likely a result of increased ER stress. The mechanism by which STZ increases Akt phosphorylation, however, remains unknown.

An O-GlcNAcase-Specific Inhibitor and Substrate Engineered by the Extension of the N-Acetyl Moiety

A novel analogue of PUGNAc, a potent O-GlcNAcase inhibitor, was synthesized and analyzed as an inhibitor of O-GlcNAcase, hexosaminidase A, and hexosaminidase B. While PUGNAc does not demonstrate selective inhibition of these related enzymes, the extension of the acetyl moiety to the longer butyl chain provided a compound with depressed inhibition of O-GlcNAcase and no observed inhibition of either hexosaminidase A or hexosaminidase B. Further, we applied this knowledge of substrate recognition at the N-acetyl group to our recently reported fluorogenic substrate for monitoring O-GlcNAcase activity. Gratifyingly, this altered small molecule was demonstrated to be a potent substrate for O-GlcNAcase while possessing no activity at hexosaminidase A. This reagent provides, for the first time, a means for monitoring O-GlcNAcase activity independent of the related enzymes hexosaminidase A and hexosaminidase B.

Inhibition of O-GlcNAcase by PUGNAc is dependent upon the oxime stereochemistry

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.