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 a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.

Discovery of MK-8768, a Potent and Selective mGluR2 Negative Allosteric Modulator.

Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

Intracranial administration of alpha-synuclein fibrils in A30P-synuclein transgenic mice causes robust synucleinopathy and microglial induction.

Synucleinopathies are neurodegenerative disorders involving pathological alpha-synuclein (αSyn) protein, including dementia with Lewy bodies, multiple system atrophy and Parkinson's disease (PD). Current in vivo models of synucleinopathy include transgenic mice overexpressing αSyn variants and methods based on administration of aggregated, exogenous αSyn. Combining these techniques offers the ability to study consequences of introducing pathological αSyn into primed neuronal environments likely to develop synucleinopathy. Herein, we characterize the impacts pre-formed fibrils (PFFs) of recombinant, human αSyn have in mice overexpressing human A30P αSyn, a mutation associated with autosomal dominant PD. A30P mouse brain contains detergent insoluble αSyn biochemically similar to PD brain, and these mice develop Lewy-like synucleinopathy with age. Administration of PFFs in A30P mice resulted in regionally-specific accumulations of phosphorylated synuclein, microglial induction and a motor phenotype that differed from PFF-induced effects in wildtype mice. Surprisingly, PFF-induced losses of tyrosine hydroxylase were similar in A30P and wildtype mice. Thus, the PFF-A30P model recapitulates key aspects of synucleinopathy with induction of microglia, creating an appropriate system for evaluating neurodegenerative therapeutics.

Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer's disease.

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.

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.

Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

Solid-phase extraction enhances detection of beta-amyloid peptides in plasma and enables Abeta quantification following passive immunization with Abeta antibodies.

We have previously developed a solid-phase extraction (SPE) procedure to enable the detection of beta-amyloid (Abeta) peptides in brain tissue from non-transgenic animals. We have now adapted these methods to enrich the Abeta fraction in cerebrospinal fluid (CSF) and plasma. Human CSF and plasma and Tg2576 mouse plasma were subjected to guanidine denaturation followed by SPE in 96-well cassettes. The resulting eluates could be concentrated significantly to enhance detection of low-abundance Abeta peptides by immunoassay. The concentrated eluates diluted in a linear fashion with consistent recovery between SPE columns. This technique was therefore used to facilitate quantification of Abeta1-X, 1-40, 1-42, and 1-38 peptides in normal human CSF and plasma samples. SPE sample preparation was also applied to the plasma of mice dosed peripherally with a monoclonal antibody raised against Abeta. When such samples were assayed directly, the presence of the systemically administered antibody interfered with the subsequent immunoassay, by preventing detection of antibody-bound Abeta. After subjecting plasma from antibody-treated animals to denaturation and SPE, the antibody-antigen complex was disrupted, and the Abeta fraction could be isolated from the antibody-containing fraction. Application of this method allowed for detection of a 100-fold increase in plasma Abeta1-40 following treatment of Tg2576 mice or wild type littermate control mice with Abeta40-specific monoclonal antibody 9TL. Given the availability of a variety of SPE matrices, we hypothesize that these methods could facilitate plasma antigen retrieval using multiple therapeutic antibody approaches.

Early intervention of tau pathology prevents behavioral changes in the rTg4510 mouse model of tauopathy.

Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.

Demonstration of a common artifact in immunosorbent assays of brain extracts: development of a solid-phase extraction protocol to enable measurement of amyloid-beta from wild-type rodent brain.

In the process of developing species-specific, immunosorbent assays for brain amyloid-beta (Abeta) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Abeta in this assay format. Using synthetic peptides, cerebrospinal fluid (CSF), or plasma samples, no nonspecific binding or cross-species immunoreactivity was detected in human or rodent Abeta assays. However, extracts of guinea pig brain (human Abeta sequence) or rat brain (rodent Abeta sequence) demonstrated immunoreactivity regardless of which capture antibody, detection antibody, or reporter method (colorimetric or fluorescent) was used. This immunoreactivity remained even in the absence of a capture antibody. Various blocking conditions failed to resolve the nonspecific binding of detection antibodies in the presence of brain extracts. Fractionation of DEA-extracted guinea pig brain over Sephadex G-50 demonstrated the feasibility of separating specific from nonspecific binding components in the brain extracts. Thus, a solid phase extraction method, compatible with multiple extraction buffers, has been developed to isolate and concentrate Abeta from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Abeta peptides in extracts from wild-type animals.

Detecting tau in serum of transgenic animal models after tau immunotherapy treatment.

In the attempt to elucidate if the "peripheral sink hypothesis" could be a potential mechanism of action for tau removal in passive immunotherapy experiments, we have examined tau levels in serum of chronically injected JNPL3 and Tg4510 transgenic animals. Measurement of tau in serum of mice treated with tau antibodies is challenging because of the antibody interference in sandwich enzyme-linked immunosorbent assays. To address this issue, we have developed a heat-treatment protocol at acidic pH to remove interfering molecules from serum, with excellent recovery of tau. The present data show that pan-tau and conformational antibodies do increase tau in mouse sera. However, these concentrations in serum do not consistently correlate with reductions of tau pathology in brain, suggesting that large elevations of tau species measured in serum are not predictive of efficacy. Here, we describe a reliable method to detect tau in serum of transgenic animals that have undergone tau immunotherapy. Levels of tau in human serum are less than the sensitivity of current assays, although artifactual signals are common. The method may be useful in similarly treated humans, a situation in which false positive signals are likely.

Tau protein phosphorylation as a therapeutic target in Alzheimer's disease.

Neurofibrillary tangles (NFTs) are a distinguishing neuropathological feature found in postmortem brains of Alzheimer s disease (AD) and tauopathy patients. The density of these lesions correlates with severity of AD and their distribution follows a characteristic pattern of expansion as the disease progresses. The principle components of NFTs are highly phosphorylated forms of the microtubule-associated protein, tau. Tau phosphorylation is believed to initiate or facilitate dissociation from microtubules leading to microtubule destabilization, decay of cellular transport properties, and cell death. This review summarizes recent data and prevailing views on the roles of protein kinases and phosphatases in the regulation of tau phosphorylation in vitro and in vivo, taking into account data from human neurodegenerative diseases and from transgenic rodent models. Small molecule inhibitors of tau phosphorylation that serve as important research tools and possibly the basis of potential new therapeutics, are also described. Key challenges in developing effective therapeutic agents include identification of the relevant kinase(s) responsible for aberrant tau phosphorylation in AD, synthesis of inhibitors selectively targeting those kinases and establishment of appropriate animal models.

Cdk5 as a drug target for the treatment of Alzheimer's disease.

Cyclin-dependent kinase-5 (cdk5) is suggested to play a role in tau phosphorylation and contribute to the pathogenesis of Alzheimer's disease (AD). One of its activators, p25, is dramatically increased in AD brains where p25 and cdk5 are colocalized with neurofibrillary tangles. Several animal models have shown a correlation of p25/cdk5 activities with tau phosphorylation. Overexpression of p25/cdk5 in nueronal cultures not only leads to tau phosphorylation but also cytoskeletal abnormalities and neurodegeneration. Therefore, cdk5 kinase inhibitors are potential therapeutic agents for the treatment of AD. Availability of potent, selective, brain permeable cdk5 inhibitors and relevant animal models in which their efficacy can be treated will be critical in the development of these inhibitors.

Avian and Human Homologues of the P2Y1 Receptor: Pharmacological, Signaling, and Molecular Properties.

[Table: see text] The P2Y receptor on turkey erythrocyte membranes was the first P2 receptor to be shown to activate phospholipase C (PLC) in a strictly guanine nucleotide-dependent manner and remains the only G protein-coupled P2 receptor for which G protein-coupling kinetics have been defined. This membrane receptor has provided a model system for detailed pharmacological analyses of a series of chain-extended 2-thioether derivatives of adenine nucleotides that exhibit remarkable selectivity and potency for P2Y receptors. This model system also has led recently to identification of a novel series of P2 receptor antagonists. The turkey erythrocyte receptor is the species homologue of the chick P2Y1 receptor originally cloned by Webb and coworkers [Webb et al., 1993]. We also have cloned the human homologue of the P2Y1 receptor, which exhibits identical pharmacological and second messenger signaling properties to that of the avian P2Y1 receptor.

Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance.

UNLABELLED: Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid accumulation and autophagic changes. Parkin is an E3 ubiquitin ligase involved in proteasomal and autophagic clearance. We previously demonstrated decreased parkin solubility and interaction with the key autophagy enzyme beclin-1 in AD, but tyrosine kinase inhibition restored parkin-beclin-1 interaction. In the current studies, we determined the mechanisms of nilotinib-induced parkin-beclin-1 interaction, which leads to amyloid clearance. Nilotinib increased endogenous parkin levels and ubiquitination, which may enhance parkin recycling via the proteasome, leading to increased activity and interaction with beclin-1. Parkin solubility was decreased and autophagy was altered in amyloid expressing mice, suggesting that amyloid stress affects parkin stability, leading to failure of protein clearance via the lysosome. Isolation of autophagic vacuoles revealed amyloid and parkin accumulation in autophagic compartments but nilotinib decreased insoluble parkin levels and facilitated amyloid deposition into lysosomes in wild type, but not parkin(-/-) mice, further underscoring an essential role for endogenous parkin in amyloid clearance. These results suggest that nilotinib boosts the autophagic machinery, leading to increased level of endogenous parkin that undergoes ubiquitination and interacts with beclin-1 to facilitate amyloid clearance. These data suggest that nilotinib-mediated autophagic changes may trigger parkin response via increased protein levels, providing a therapeutic strategy to reduce Aß and Tau in AD. KEY MESSAGE: Parkin solubility (stability) is decreased in AD and APP transgenic mice. Nilotinib-induced autophagic changes increase endogenous parkin level. Increased parkin level leads to ubiquitination and proteasomal recycling. Re-cycling decreases insoluble parkin and increases parkin-beclin-1 interaction. Beclin-1-parkin interaction enhances amyloid clearance.

High throughput monitoring of amyloid-ß(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-ß (Aß)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.

Peripheral elevation of IGF-1 fails to alter Abeta clearance in multiple in vivo models.

Increasing beta-amyloid (Abeta) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Abeta clearance by facilitating transport of Abeta out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Abeta acutely, with reduction in plaque pathology after chronic treatment. Thus Abeta levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Abeta levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Abeta. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Abeta were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Abeta (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Abeta levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor, LY-450139.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.