lrrk2-in1 and Parkinson-Disease

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

Topics: Adult; Aged; Benzodiazepinones; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Female; Fibroblasts; Fluorescence Resonance Energy Transfer; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Middle Aged; Mitochondria; Mitophagy; Mutation; Parkinson Disease; Phosphorylation; Primary Cell Culture; Protein Kinases; Pyrimidines; Ubiquitin-Protein Ligases

LRRK2-IN-1, one of the first selective inhibitors of leucine-rich repeat kinase 2 (LRRK2), was serendipitously found to exhibit potent antiproliferative activity in several types of human cancer cells. In this study, we employed a chemoproteomic strategy utilizing a photoaffinity probe to identify the cellular target(s) of LRRK2-IN-1 underlying its anticancer activity. LRRK2-IN-1 was found to induce cell cycle arrest as well as cancer cell death by specifically binding to human proliferating cell nuclear antigen (PCNA) in cancer cells. Our current findings suggest the potential of LRRK2-IN-1 as a novel pharmacological molecule for scrutinizing cell physiology and furnish a logical foundation for the future development of therapeutic reagents for cancer.

Topics: Antiparkinson Agents; Benzodiazepinones; Cell Proliferation; Drug Repositioning; Drug Screening Assays, Antitumor; Humans; Inhibitory Concentration 50; Jurkat Cells; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Molecular Probes; Parkinson Disease; Photoaffinity Labels; Proliferating Cell Nuclear Antigen; Protein Kinase Inhibitors; Proteomics; Pyrimidines

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent cause of familial and sporadic Parkinson's disease (PD). Because most pathogenic LRRK2 mutations result in enhanced kinase activity, it suggests that LRRK2 inhibitors may serve as a potential treatment for PD. To evaluate whether LRRK2 inhibitors are effective therapies for PD, it is crucial to know whether LRRK2 inhibitors will affect dopaminergic (DAergic) neurotransmission. However, to date, there is no study to investigate the impact of LRRK2 inhibitors on DAergic neurotransmission.. To address this gap in knowledge, we examined the effects of three types of LRRK2 inhibitors (LRRK2-IN-1, GSK2578215A, and GNE-7915) on dopamine (DA) release in the dorsal striatum using fast-scan cyclic voltammetry and DA neuron firing in the substantia nigra pars compacta (SNpc) using patch clamp in mouse brain slices.. We found that LRRK2-IN-1 at a concentration higher than 1 μM causes off-target effects and decreases DA release, whereas GSK2578215A and GNE-7915 do not. All three inhibitors at 1 μM have no effect on DA release and DA neuron firing rate. We have further assessed the effects of the inhibitors in two preclinical LRRK2 mouse models (i.e., BAC transgenic hG2019S and hR1441G) and demonstrated that GNE-7915 enhances DA release and synaptic vesicle mobilization/recycling.. GNE-7915 can be validated for further therapeutic development for PD.

Topics: Aminopyridines; Animals; Benzamides; Benzodiazepinones; Biophysical Phenomena; Corpus Striatum; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Mice; Mice, Transgenic; Morpholines; Mutation; Parkinson Disease; Patch-Clamp Techniques; Pyrimidines; Substantia Nigra

It has been uncertain whether specific disease-relevant biomarker phenotypes can be found using sporadic Parkinson's disease (PD) patient-derived samples, as it has been proposed that there may be a plethora of underlying causes and pathological mechanisms. Fibroblasts derived from familial PD patients harboring leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and Parkin mutations show clear disease-relevant mitochondrial phenotypes, which are exacerbated under conditions of pharmacological stress. We utilized fibroblasts derived from non-familial sporadic PD patients (without LRRK2 mutations) or LRRK2 mutation carriers to directly compare the cellular phenotypes during and after mitochondrial stress. We then determined the effects of pharmacological LRRK2 kinase inhibition using LRRK2-in-1. We found that there were two distinct populations of sporadic PD patient-derived fibroblast lines. One group of sporadic PD lines was highly susceptible to valinomycin-induced mitochondrial depolarization, emulating the mutant LRRK2 phenotype. These lines showed elevated mitochondrial superoxide/ nitric oxide levels, displayed increased mitochondrial and lysosome co-localization, and an increased rate of mitochondrial collapse, which corresponded with changes in mitochondrial fission and fusion proteins. The application of LRRK2-in-1 reversed decreased levels of mitochondrial and lysosome co-localization and partially restored mitochondrial network associated proteins and the mitochondrial membrane potential in the fibroblasts. This study identifies novel mitochondrial biomarkers in sporadic PD patient-derived fibroblast lines, which could be used as preclinical tools in which to test novel and known neuroprotective compounds.

Topics: Benzodiazepinones; Biomarkers; Cell Line; Fibroblasts; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Dynamics; Parkinson Disease; Protein Kinases; Pyrimidines; Stress, Physiological; Ubiquitin-Protein Ligases; Valinomycin

There is an urgent need for the development of Parkinson's disease (PD) treatments that can slow disease progression. The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to PD, and modulation of LRRK2 enzymatic activity has been proposed as a novel therapeutic strategy. In this review, we describe the bioactivity of selected small molecules that have been used to inhibit LRRK2 kinase activity in vitro or in vivo. These compounds are important tools for understanding the cellular biology of LRRK2 and for evaluating the potential of LRRK2 inhibitors as disease-modifying PD therapies.

Topics: Animals; Antiparkinson Agents; Brain; Guanosine Triphosphate; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mutation; Parkinson Disease; Permeability; Protein Binding; Protein Conformation; Protein Serine-Threonine Kinases

LRRK2 (leucine-rich repeat protein kinase 2) is one of the most commonly accepted genes associated with Parkinson's disease (PD). The overexpression of disease-associated mutations in LRRK2 is toxic to the cells, while reduction or elimination of LRRK2 expression promotes cell health and growth. Thus, the identification of an LRRK2 inhibitor with good physiochemical and pharmacokinetic properties is of great interest for the treatment of PD.. In this study, we have investigated LRRK2 compounds, LRRK2-IN-1 and Compound 1, in vitro and in vivo to determine how suitable they are as a selective LRRK2 tool compound.. We report that Compound 1, patented by GSK, is a potent and selective LRRK2 inhibitor with good blood-brain barrier permeability as reflected by its high brain to plasma ratio in rats. In addition, Compound 1 can significantly promote neurite outgrowth in a primary cortical culture, indicating an optimistic cellular function of this compound in a biological system. In contrast, LRRK2-IN-1 is a less selective LRRK2 inhibitor and has low brain penetration. Furthermore, LRRK2-IN-1 is cyto- and genotoxic, while Compound 1 does not exhibit any toxicity.. These results suggest that Compound 1 may be a superior tool compound than LRRK2-IN-1 to advance future pharmacological research on LRRK2.

Topics: Animals; Benzodiazepinones; Blood-Brain Barrier; Caco-2 Cells; Cell Survival; Drug Discovery; Drug Stability; Hep G2 Cells; Hepatocytes; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Metabolic Clearance Rate; Microsomes, Liver; Parkinson Disease; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Rats, Sprague-Dawley; Substrate Specificity; Tissue Distribution

Topics: Animals; Benzodiazepinones; Drosophila; Drosophila Proteins; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mutation; Parkinson Disease; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Vision Disorders

A cluster of phosphorylation sites in LRRK2 (leucine-rich repeat kinase 2), including Ser910, Ser935, Ser955 and Ser973, is important for PD (Parkinson's disease) pathogenesis as several PD-linked LRRK2 mutants are dephosphorylated at these sites. LRRK2 is also dephosphorylated in cells after pharmacological inhibition of its kinase activity, which is currently proposed as a strategy for disease-modifying PD therapy. Despite this importance of LRRK2 dephosphorylation in mutant LRRK2 pathological mechanism(s) and in LRRK2's response to inhibition, the mechanism by which this occurs is unknown. Therefore we aimed to identify the phosphatase for LRRK2. Using a panel of recombinant phosphatases, we found that PP1 (protein phosphatase 1) efficiently dephosphorylates LRRK2 in vitro. PP1 activity on LRRK2 dephosphorylation was confirmed in cells using PP1 inhibition to reverse LRRK2 dephosphorylation induced by the potent LRRK2 kinase inhibitor LRRK2-IN1 as well as in R1441G mutant LRRK2. We also found that PP1 and LRRK2 can form a complex in cells. Furthermore, we observed that PP1 inhibition modulates LRRK2's cellular phenotype by reducing skein-like LRRK2-positive structures associated with dephosphorylation. In conclusion, the present study reveals PP1 as the physiological LRRK2 phosphatase, responsible for LRRK2 dephosphorylation observed in PD mutant LRRK2 and after LRRK2 kinase inhibition.

Topics: Animals; Benzodiazepinones; Cell Line; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Mutation; Parkinson Disease; Phosphorylation; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Pyrimidines

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Amino Acid Sequence; Amino Acid Substitution; Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Benzodiazepinones; Binding, Competitive; Enzyme Activation; Epitopes; HEK293 Cells; Humans; Immunoprecipitation; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Mice, Inbred C57BL; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Parkinson Disease; Phosphorylation; Protein Binding; Protein Conformation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Pyrimidines; Recombinant Proteins; Swiss 3T3 Cells

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.

Topics: Animals; Brain; Cell Line; Cells, Cultured; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Models, Molecular; Mutation; Parkinson Disease; Protein Serine-Threonine Kinases; Pyrimidines

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson's disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10 nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0 μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.

Topics: 3T3 Cells; Animals; Brain; Cell Line; Drug Discovery; HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Mice; Models, Molecular; Mutation; Parkinson Disease; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Structural Homology, Protein

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. We employed a new, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor, LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910 and Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson's disease.

Topics: Benzodiazepinones; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mutation; Parkinson Disease; Phosphorylation; Protein Serine-Threonine Kinases; Pyrimidines; Serine