Fluorinated Isoindolinone-Based Glucosylceramide Synthase Inhibitors with Low Human Dose Projections.

Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead , we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution.

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).

Pyrazole Ureas as Low Dose, CNS Penetrant Glucosylceramide Synthase Inhibitors for the Treatment of Parkinson's Disease.

Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS).

A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function.

Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD.

LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson's disease.

LRRK2 has been implicated in endolysosomal function and likely plays a central role in idiopathic Parkinson's disease (iPD). In iPD, dopaminergic neurons within the substantia nigra are characterized by increased LRRK2 kinase activity, endolysosomal deficits, and accumulation of autophagic vesicles with incompletely degraded substrates, including α-synuclein. Although LRRK2 has been implicated in endolysosomal and autophagic function, it remains unclear whether inhibition of LRRK2 kinase activity can prevent endolysosomal deficits or reduce dopaminergic neurodegeneration.

Functionalization of the TMEM175 p.M393T variant as a risk factor for Parkinson disease.

Multiple genome-wide association studies (GWAS) in Parkinson disease (PD) have identified a signal at chromosome 4p16.3; however, the causal variant has not been established for this locus. Deep investigation of the region resulted in one identified variant, the rs34311866 missense SNP (p.

Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis.

TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation.

Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease.

Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors.

Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection.

Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells.

Unlabelled: Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau.

Non-motor parkinsonian pathology in aging A53T α-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function.

Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α-synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH).

Leucine-rich repeat kinase 2 (LRRK2) cellular biology: a review of recent advances in identifying physiological substrates and cellular functions.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common forms of inheritable Parkinson's disease and likely play a role in sporadic disease as well. LRRK2 is a large multidomain protein containing two key groups, a Ras-like GTP binding domain and a serine, threonine kinase domain. Mutations in the LRRK2 gene that associate with Parkinson's disease reside primarily within the two functional domains of the protein, suggesting that LRRK2 function is critical to the pathogenesis of the disease.

Chronic rotenone exposure reproduces Parkinson's disease gastrointestinal neuropathology.

Gastrointestinal disorders, particularly severe constipation and delayed gastric emptying, are core symptoms of Parkinson's disease that affect most patients. However, the neuropathological substrate and physiological basis for this dysfunction are poorly defined. To begin to explore these phenomena in laboratory models of PD, rats were treated with either vehicle or rotenone (2.

A highly reproducible rotenone model of Parkinson's disease.

The systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD.

Effects of sildenafil on nigrostriatal dopamine neurons in a murine model of Parkinson's disease.

The objective of this study was to determine if the phosphodiesterase 5 (PDE-5) inhibitor, sildenafil, could be used as a neuroprotective agent in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of Parkinson's disease. The underlying hypothesis of these studies is that blockade of PDE-5 catabolism of cGMP will attenuate the loss of nigrostriatal dopamine (NSDA) neurons following chronic neurotoxin exposure. Chronic MPTP-treated mice were administered sildenafil using three different regimens.

Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism.

Many neurodegenerative diseases associated with functional Tau dysregulation, including Alzheimer's disease (AD) and other tauopathies, also show alpha-synuclein (alpha-Syn) pathology, a protein associated with Parkinson's disease (PD) pathology. Here we show that treatment of primary mesencephalic neurons (48 h) or subchronic treatment of wild-type (WT) mice with the Parkinsonism-inducing neurotoxin MPP+/MPTP, results in selective dose-dependent hyperphosphorylation of Tau at Ser396/404 (PHF-1-reactive Tau, p-Tau), with no changes in pSer202 but with nonspecific increases in pSer262 levels. The presence of alpha-Syn was absolutely mandatory to observe MPP+/MPTP-induced increases in p-Tau levels, since no alterations in p-Tau were seen in transfected cells not expressing alpha-Syn or in alpha-Syn-/- mice.

Substrate-mediated enhancement of phosphorylated tyrosine hydroxylase in nigrostriatal dopamine neurons: evidence for a role of alpha-synuclein.

Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity.

Mice lacking alpha-synuclein have an attenuated loss of striatal dopamine following prolonged chronic MPTP administration.

The functional role of alpha-synuclein in the pathogenesis of Parkinson's disease (PD) is not fully understood. Systemic exposure of alpha-synuclein-deficient mice to neurotoxins provides a direct approach to evaluate how alpha-synuclein may mediate cell death in a common murine model of PD. To this end, wild-type and homozygous alpha-synuclein knock-out mice were treated with sub-chronic and prolonged, chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).