Ernest McEachern

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 Alzheimers 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.

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

BackgroundAmyloid 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.ResultsWe 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 β-amyloid peptide levels and decreased levels of amyloid plaques.ConclusionsThis study indicates that increased O-GlcNAc can influence β-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.

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.

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

BackgroundHyperphosphorylation 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.MethodsGenetic 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.ResultsSubstantial (>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.ConclusionThe 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.

EARLY CLINICAL RESULTS AND PRECLINICAL VALIDATION OF THE O-GLCNACASE (OGA) INHIBITOR MK-8719 AS A NOVEL THERAPEUTIC FOR THE TREATMENT OF TAUOPATHIES

cant decrease of human Tau multimers (HT7/HT7 setup), as well as a significant decrease of a misfolded Tau (MC-1/HT7 setup) in a total brain fraction.A trend to decrease humanTau phosphorylated at pS396 in different brain fractions was also observed. ACI-35 treatment significantly improved the rotarod performance in both the transgenic hTauP301S-Tg model and the AAV-TauP301S spreading model. Conclusions: The study demonstrated that active immunization with ACI-35 significantly decreased human pathological Tau species in different brain fractions and ameliorated the clinical phenotype in two different mouse models of tauopathy.

PHARMACOKINETICS AND PHARMACODYNAMICS TO SUPPORT CLINICAL STUDIES OF MK-8719: AN O-GLCNACASE INHIBITOR FOR PROGRESSIVE SUPRANUCLEAR PALSY

generative disease. Stress is implicated in the development of AD since oxidative stress has been linked to cognitive impairment. Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea and has antioxidant, anti-inflammatory and anti-atherogenic effects, while Diazepam is an anxiolytic with promising neuroprotective properties. Methods: Seven groups (8 rats/ group) were daily IP injected for six week either with saline for control (2 groups) or with 70 mg/kg ALCL3 for AD-induced model (5 groups). Stress was induced for all groups except one control and one ALCL3 group by exposing rats 6 times during six weeks to Stress-induced box paradigm (1time/ week for 30 minute). Three groups of AD-induced model were also daily received either EGCG (10mg/kg, IP), Diazepam (0.1mg/kg, IP) or their combination. All rats were examined in two behavioral experiments; Morris water maze task and Conditioned-avoidance learning test. Histological examination was achieved in different brain regions and biochemical measurements as brain cholinergic markers (ACHE); oxidative stress markers (SOD, GSH-Px, MDA, TAC) and inflammatory mediators (TNF-a, IL-1b) were also assayed for all groups. Results: Rats exposed to ALCL3 together with stress showed marked decline in learning and memory abilities. Stress also induced significant elevation in hippocampus TNF-a, IL-1b andMDA level as well as in ACHE activity accompanied by reduction in GSH-Px, TAC and SOD activities. Marked histopathological brain degenerations were also shown in AD-model group exposed to stress. EGCG showed more marked protective effect than Diazepam from stress-potentiated the deleterious effect of ALCL3 on the brain, however Co-administration of both resulted in more pronounced protection as regarding all measured parameters. Conclusions:Exposure to stress represents a risk factor in induction and progression of AD. The deleterious effect of stress on the brain and hippocampus can be counteracted by Co-administration of both EGCG and Diazepam.