MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

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.

Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice.

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Structures of lactate dehydrogenase A (LDHA) in apo, ternary and inhibitor-bound forms.

Lactate dehydrogenase (LDH) is an essential metabolic enzyme that catalyzes the interconversion of pyruvate and lactate using NADH/NAD(+) as a co-substrate. Many cancer cells exhibit a glycolytic phenotype known as the Warburg effect, in which elevated LDH levels enhance the conversion of glucose to lactate, making LDH an attractive therapeutic target for oncology. Two known inhibitors of the human muscle LDH isoform, LDHA, designated 1 and 2, were selected, and their IC50 values were determined to be 14.4 ± 3.77 and 2.20 ± 0.15 µM, respectively. The X-ray crystal structures of LDHA in complex with each inhibitor were determined; both inhibitors bind to a site overlapping with the NADH-binding site. Further, an apo LDHA crystal structure solved in a new space group is reported, as well as a complex with both NADH and the substrate analogue oxalate bound in seven of the eight molecules and an oxalate only bound in the eighth molecule in the asymmetric unit. In this latter structure, a kanamycin molecule is located in the inhibitor-binding site, thereby blocking NADH binding. These structures provide insights into LDHA enzyme mechanism and inhibition and a framework for structure-assisted drug design that may contribute to new cancer therapies.

Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice.

BACKGROUND: Amyloid plaques and neurofibrillary tangles (NFTs) are the defining pathological hallmarks of Alzheimer's disease (AD). Increasing the quantity of the O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of nuclear and cytoplasmic proteins slows neurodegeneration and blocks the formation of NFTs in a tauopathy mouse model. It remains unknown, however, if O-GlcNAc can influence the formation of amyloid plaques in the presence of tau pathology. RESULTS: We treated double transgenic TAPP mice, which express both mutant human tau and amyloid precursor protein (APP), with a highly selective orally bioavailable inhibitor of the enzyme responsible for removing O-GlcNAc (OGA) to increase O-GlcNAc in the brain. We find that increased O-GlcNAc levels block cognitive decline in the TAPP mice and this effect parallels decreased ß-amyloid peptide levels and decreased levels of amyloid plaques. CONCLUSIONS: This study indicates that increased O-GlcNAc can influence ß-amyloid pathology in the presence of tau pathology. The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease.

Discovery of novel small molecule orally bioavailable C-X-C chemokine receptor 4 antagonists that are potent inhibitors of T-tropic (X4) HIV-1 replication.

The redesign of azamacrocyclic CXCR4 chemokine receptor antagonists resulted in the discovery of novel, small molecule, orally bioavailable compounds that retained T-tropic (CXCR4 using, X4) anti-HIV-1 activity. A structure-activity relationship (SAR) was determined on the basis of the inhibition of replication of X4 HIV-1 NL4.3 in MT-4 cells. As a result of lead optimization, we identified (S)-N'-((1H-benzo[d]imidazol-2-yl)methyl)-N'-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-diamine (AMD070) 2 as a potent and selective antagonist of CXCR4 with an IC(50) value of 13 nM in a CXCR4 125I-SDF inhibition binding assay. Compound 2 inhibited the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC(50) of 2 and 26 nM, respectively, while remaining noncytotoxic to cells at concentrations exceeding 23 microM. The pharmacokinetics of 2 was evaluated in rat and dog, and good oral bioavailability was observed in both species. This compound represents the first small molecule orally bioavailable CXCR4 antagonist that was developed for the treatment of HIV-1 infection.

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

Asymmetric synthesis of oxygen heterocycles via Pd-catalyzed dynamic kinetic asymmetric transformations: application to nucleosides.

Racemic butadiene and isoprene monoepoxide react with unsaturated alcohols in the presence of a chiral palladium catalyst and a boron co-catalyst to give 3-alkoxy-4-hydroxy-1-butene and 3-alkoxy-4-hydroxy-3-methyl-1-butene, respectively, with excellent regio- and enantioselectivity in a dynamic kinetic asymmetric transformation whereby both enantiomers of the starting epoxides provide the same enantiomeric product. In the case of 2-phenylbutadiene monoepoxide, easily available from phenacyl chloride and vinylmagnesium bromide, the reaction proceeds by kinetic resolution. A model to rationalize the result is presented. The bis-olefin products are ideal substrates for the Ru catalyzed ring closing metathesis. In this way, five-, six-, and seven-membered oxygen heterocycles are readily available enantiomerically pure. The value of this very simple two step process is demonstrated by the use of the five-membered ring heterocycles to form unnatural and unusual nucleosides that cannot be easily accessed by other means. The sequence involves a Ru catalyzed isomerization of the initial 2,5-dihydrofuran to a 2,3-dihydrofuran followed by a selenium promoted addition of a pyrimidine or purine base. One advantage of this strategy is the easy access to either enantiomeric series, both of which have important biological applications.

Enzymatic resolution of bicyclic 1-heteroarylamines using Candida antarctica lipase B.

Candida antarctica lipase B has been used to kinetically resolve a structurally diverse series of bicyclic 1-heteroaryl primary amines by enantioselective acetylation. High yields of either enantiomer could be obtained with excellent enantioselectivity (90-99% ee), while the undesired enantiomer could, in some cases, be recycled by thermal racemization. The absolute stereochemistry of the products was confirmed by an X-ray crystal structure.

Concise preparation of amino-5,6,7,8-tetrahydroquinolines and amino-5,6,7,8-tetrahydroisoquinolines via catalytic hydrogenation of acetamidoquinolines and acetamidoisoquinolines.

A method to prepare amino-substituted 5,6,7,8-tetrahydroquinolines and 5,6,7,8-tetrahydroisoquinolines via catalytic hydrogenation of the corresponding acetamido-substituted quinolines and isoquinolines followed by acetamide hydrolysis is described. The yields of the products are good when the acetamido substituent is present on the pyridine ring and moderate with the acetamido substituent on the benzene ring. This method has also been applied to the regioselective reduction of quinoline substrates bearing other substituents (R = OMe, CO(2)Me, Ph).