Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond

The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson’s disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases. Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2 kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective inhibitor of the present series, is characterized by an IC₅₀ of 15 nM against wild-type LRRK2 and 10 nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled toxicity determination in whole organisms.     

Discovery of LRRK2 inhibitors by using an ensemble of virtual screening methods

In this paper, we present the results of a ligand- and structure-based virtual screen targeting LRRK2, a kinase that has been implicated in Parkinson’s disease. For the ligand-based virtual screen, the structures of 12 competitor compounds were used as queries for a variety of 2D and 3D searches. The structure-based virtual screen relied on homology models of LRRK2, as no X-ray structure is currently available in the public domain. From the virtual screening, 662 compounds were purchased, of which 35 showed IC₅₀ values below 10 μM in wild-type and/or mutant LRRK2 (a hit rate of 5.3%). Of these 35 hits, four were deemed to have potential for medicinal chemistry follow-up.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.     

ULK1 and JNK are involved in mitophagy incurred by LRRK2 G2019S expression

Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson’s disease (PD). We and others reported recently that expression of the pathogenic gainoffunction mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fi ssion in neurons through DLP1. Here we provide evidence that expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fi ssion through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our fi ndings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.     

Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent,selective and orally bioavailable LRRK2 inhibitors

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson’s disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100?mg/kg) and mice (45?mg/kg) following oral administration.     

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.     

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.     

The design and SAR of a novel series of 2-aminopyridine based LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson’s disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.     

5-Substituted-N-pyridazinylbenzamides as potent and selective LRRK2 inhibitors: Improved brain unbound fraction enables efficacy

We describe the discovery and optimization of 5-substituted-N-pyridazinylbenzamide derivatives as potent and selective LRRK2 inhibitors. Extensive SAR studies led to the identification of compounds 18 and 23, which demonstrated good in vitro pharmacokinetic profile and excellent selectivity over 140 other kinases. Both compounds demonstrated high unbound fractions in both blood and brain. Compound 18 proved to be brain penetrant, and the high unbound fraction of compound 18 in brain enabled its in vivo efficacy in CNS, wherein a significant inhibition of LRRK2 Ser935 phosphorylation was observed in rat brain following intravenous infusion at 5?mg/kg/h.     

Differential protein–protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1‐ and LRRK2‐specific cellular processes by identifying their distinct interacting proteins. A protein microarray‐based interaction screen was performed with recombinant 3xFlag‐LRRK1 and 3xFlag‐LRRK2 and, in parallel, co‐immunoprecipitation followed by mass spectrometry was performed from SH‐SY5Y neuroblastoma cell lines stably expressing 3xFlag‐LRRK1 or 3xFlag‐LRRK2. We identified a set of LRRK1‐ and LRRK2‐specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF‐R) as a LRRK1‐specific interactor, while 14‐3‐3 proteins were LRRK2‐specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14‐3‐3 consensus binding motifs. To assess the functional relevance of these interactions, SH‐SY5Y‐LRRK1 and ‐LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14‐3‐3 binding, or with EGF, an EGF‐R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins.

     

Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. An amino terminal cluster of constitutively phosphorylated residues, serines 860, 910, 935, 955, and 973, appears to be biologically relevant. Phosphorylation of serines 910 and 935 is regulated in response to LRRK2 kinase activity and is responsible for interaction with 14-3-3 and maintaining LRRK2 in a non-aggregated state. We examined the phosphorylation status of two other constitutive phosphorylation sites, serines 955 and 973. Treatment of LRRK2 expressing cells with the selective LRRK2 inhibitor LRRK2-IN1 revealed that, like Ser910/Ser935, phosphorylation of Ser955 and Ser973 is disrupted by acute inhibition of LRRK2 kinase activity. Additionally, phosphorylation of Ser955 and 973 is disrupted in the context of several Parkinson's disease associated mutations [R1441G/C, Y1699C, and I2020T]. We observed that modification of Ser973 is dependent on the modification of Ser910/Ser935. Ser955Ala and Ser973Ala mutations do not induce relocalization of LRRK2; however, all phosphomutants exhibited similar localization patterns when exposed to LRRK2-IN1. We conclude that the mechanisms of regulation of Ser910/935/955/973 phosphorylation are similar and physiologically relevant. These sites can be utilized as biomarkers for LRRK2 activity as well as starting points for the elucidation of upstream and downstream enzymes that regulate LRRK2.     

The development of CNS-active LRRK2 inhibitors using property-directed optimisation

Mutations in PARK8/LRRK2 are the most common genetic cause of Parkinson’s disease. Inhibition of LRRK2 kinase activity has neuroprotective benefits, and provides a means of addressing the underlying biochemical cause of Parkinson’s disease for the first time. Initial attempts to develop LRRK2 inhibitors were largely unsuccessful and highlight shortcomings intrinsic to traditional, high throughput screening methods of lead discovery. Recently, amino-pyrimidine GNE-7915 was reported as a potent (IC₅₀ = 9 nM) selective (1/187 kinases), brain-penetrant and non-toxic inhibitor of LRRK2. The use of in silico modelling, extensive in vitro assays and resource-efficient in vivo techniques to produce GNE-7915, reflects a trend towards the concerted optimisation of potency, selectivity and pharmacokinetic properties in early-stage drug development.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species     

Structural analysis of the full-length human LRRK2

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Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinson's disease (PD), an age-dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. Here, we show that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP), a negative regulator of eIF4E-mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E-BP pathway by LRRK2 stimulates eIF4E-mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. Our results suggest that chronic inactivation of 4E-BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age-dependent loss of DA neurons.     

nhe regulation and deregulation of Wnt signaling by PARK genes in health and disease

Wingless/Int （Wnt） signaling pathways are signal transduction mechanisms that have been widely studied In the field of embryogen- esis. Recent work has established a critical role for these pathways in brain development, especially of midbrain dopaminergic neu- rones, However, the fundamental importance of Wnt signaling for the normal function of mature neurones in the adult central nervous system has also lately been demonstrated by an increasing number of studies. Parkinson＇s disease （PD） is the second most prevalent neurodegenerative disease worldwide and is currently incurable. This debilitating disease is characterized by the progres- sive loss of a subset of midbrain dopaminergic neurones in the substontla nigm leadingto typical extrapyramidal motor symptoms. The aetiology of PD is poorly understood but work performed over the Last two decades has identified a growing number of genetic defects that underlie this condition. Herewe review a growing body of data connecting genes implicated in PD--most notablythe PARKgenes-- with Wnt signaling. These observations provide clues to the normal function of these proteins in healthy neurones and suggest that deregulated Wnt signaling might be a frequent pathomechanlsm leading to PD. These observations have implications for the patho- genesis and treatment of neurodegenerative diseases in general.