Structure of the Roc-COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase

Correction to: The EMBO Journal (2008) 27, 2239-2249. doi:10.1038/emboj.2008.150.     

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.     

Structural analysis of the full-length human LRRK2

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The Parkinson disease-associated protein kinase LRRK2 exhibits MAPKKK activity and phosphorylates MKK3/6 and MKK4/7, in vitro

Autosomal dominant mutations in the human Leucine-Rich Repeat Kinase 2 ( LRRK2 ) gene represent the most common monogenetic cause of Parkinson disease (PD) and increased kinase activity observed in pathogenic mutants of LRRK2 is most likely causative for PD-associated neurotoxicity. The sequence of the LRRK2 kinase domain shows similarity to MAP kinase kinase kinases. Furthermore, LRRK2 shares highest sequence homology with mixed linage kinases which act upstream of canonical MAPKK and are involved in cellular stress responses. Therefore, we addressed the question if LRRK2 exhibits MAPKKK activity by systematically testing MAPKKs as candidate substrates, in vitro . We demonstrate that LRRK2 variants phosphorylate mitogen-activated protein kinase kinases (MAPKK), including MKK3 -4, -6 and -7. MKKs act upstream of the MAPK p38 and JNK mediating oxidative cell stress, neurotoxicity and apoptosis. The disease-associated LRRK2 G2019S and I2020T mutations show an increased phosphotransferase activity towards MKKs correlating with the activity shown for its autophosphorylation. Our findings present evidence of a new class of molecular targets for mutant LRRK2 that link to neurotoxicity, cellular stress, cytoskeletal dynamics and vesicular transport.     

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.     

Identification of chemicals to inhibit the kinase activity of leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated protein

Parkinson’s disease (PD) is a late-onset neurodegenerative disease which occurs at more than 1% in populations aging 65-years and over. Recently, leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for autosomal dominantly inherited familial PD cases. LRRK2 G2019S which is a prevalent mutant found in familial PD patients with LRRK2 mutations, exhibited kinase activity stronger than that of the wild type, suggesting the LRRK2 kinase inhibitor as a potential PD therapeutics. To develop such therapeutics, we initially screened a small chemical library and selected compound 1, whose IC₅₀ is about 13.2 μM. To develop better inhibitors, we tested five of the compound 1 derivatives and found a slightly better inhibitor, compound 4, whose IC₅₀ is 4.1 μM. The cell-based assay showed that these two chemicals inhibited oxidative stress-induced neurotoxicity caused by over-expression of a PD-specific LRRK2 mutant, G2019S. In addition, the structural analysis of compound 4 suggested hydrogen bond interactions between compound 4 and Ala 1950 residue in the backbone of the ATP binding pocket of LRRK2 kinas domain. Therefore, compound 4 may be a promising lead compound to further develop a PD therapeutics based on LRRK2 kinase inhibition.     

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.     

Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2—Discovery of LRRK2 inhibitors

LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.     

Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson’s disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand–protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.     

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.     

LRRK2 as a modulator of lysosomal calcium homeostasis with downstream effects on autophagy

Alterations in autophagy are thought to underlie various neurodegenerative diseases including Parkinson disease (PD). Previous studies have indicated that the PD gene leucine rich repeat kinase 2 (LRRK2) is involved in this process, but its mechanism of action has remained unknown. Our recent work describes how LRRK2 acts through calcium-mediated events originating from acidic stores to regulate autophagy and cell survival, which may give rise to novel therapeutic strategies.     

Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity

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Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain¹. The discovery in 2004 of mutations in LRRK2 that cause Parkinson''s disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state^2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities^4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations^7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool^9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson''s is open to debate.A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads^4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK2¹². Recently, Daniëls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available¹³. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP^14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.     

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.     

Design,synthesis, and evaluation of a selective chemosensor for leucine-rich repeat kinase 2

We describe the design, synthesis, and evaluation of a selective activity probe for leucine-rich repeat kinase 2 (LRRK2), a possible molecular target for the treatment of Parkinson’s disease. Our optimal chemosensor design, termed Nictide-S2, incorporates a phosphorylation-sensitive sulfonamido-oxine fluorophore at an engineered cysteine within the substrate sequence. This design allows for the direct, real-time analysis of LRRK2 kinase activity with a detection limit of 2.5 nM. Under optimized conditions, we measured a Z′ factor of 0.7 demonstrating the potential utility of this assay for inhibitor screening. Off-target kinases capable of phosphorylating Nictide-S2 are identified and an optimized inhibitor cocktail for suppressing background signal is provided. The resulting chemosensor could be utilized to identify LRRK2 inhibitors as well as selectively report on LRRK2 activity in the presence of off-target kinases.     

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.     

Regulatory imbalance between LRRK2 kinase,PPM1H phosphatase,and ARF6 GTPase disrupts the axonal transport of autophagosomes

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