Leucine-rich Repeat Kinase 2 Inhibitors Research Articles

Structural Insights and Development of LRRK2 Inhibitors for Parkinson's Disease in the Last Decade.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, characterized by the specific loss of dopaminergic neurons in the midbrain. The pathophysiology of PD is likely caused by a variety of environmental and hereditary factors. Many single-gene mutations have been linked to this disease, but a significant number of studies indicate that mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a potential therapeutic target for both sporadic and familial forms of PD. Consequently, the identification of potential LRRK2 inhibitors has been the focus of drug discovery. Various investigations have been conducted in academic and industrial organizations to investigate the mechanism of LRRK2 in PD and further develop its inhibitors. This review summarizes the role of LRRK2 in PD and its structural details, especially the kinase domain. Furthermore, we reviewed in vitro and in vivo findings of selected inhibitors reported to date against wild-type and mutant versions of the LRRK2 kinase domain as well as the current trends researchers are employing in the development of LRRK2 inhibitors.

Recent Update on the Development of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors: A Promising Target for the Treatment of Parkinson's Disease.

Parkinson's disease is a relatively common neurological disorder with incidence increasing with age. Since current medications only relieve the symptoms and do not change the course of the disease, therefore, finding disease-modifying therapies is a critical unmet medical need. However, significant progress in understanding how genetics underpins Parkinson's disease (PD) has opened up new opportunities for understanding disease pathogenesis and identifying possible therapeutic targets. One such target is leucine-rich repeat kinase 2 (LRRK2), an elusive enzyme implicated in both familial and idiopathic PD risk. As a result, both academia and industry have promoted the development of potent and selective inhibitors of LRRK2. In this review, we have summarized recent progress in the discovery and development of LRKK2 inhibitors as well as the bioactivity of several small-molecule LRRK2 inhibitors that have been used to inhibit LRRK2 kinase activity in vitro or in vivo.

LRRK2 kinase activity regulates GCase level and enzymatic activity differently depending on cell type in Parkinson’s disease

Leucine-rich repeat kinase 2 (LRRK2) is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways, and immune function. Mutations in LRRK2 cause autosomal-dominant forms of Parkinson’s disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we performed a comprehensive analysis of GCase levels and activity in complementary LRRK2 models, including (i) LRRK2 G2019S knock in (GSKI) mice, (ii) peripheral blood mononuclear cell (PBMCs), plasma, and fibroblasts from PD patients carrying LRRK2 G2019S mutation, (iii) patient iPSCs-derived neurons; (iv) endogenous and overexpressed cell models. In some of these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. GCase protein level is reduced in GSKI brain tissues and in G2019S iPSCs-derived neurons, but increased in fibroblasts and PBMCs from patients, suggesting cell-type-specific effects. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase in a cell-type-specific manner, with important implications in the context of therapeutic application of LRRK2 inhibitors in GBA1-linked and idiopathic PD.

Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

LRRK‐2 Inhibition By PF06447475 Treatment Reduces Neuronal Damage and Immune Response After Spinal Cord Trauma

Spinal Cord Injury (SCI) is a devastating event followed by neurodegeneration, activation of the inflammatory cascade and immune system. Dysregulated or non‐resolving inflammatory processes can affect neuronal homeostasis and drive immune cells stimulation. The leucine‐rich‐repeat kinase 2 (LRRK2) is a gene associated with the progression of Parkinson’s disease (PD), and its kinase activity was found to be upregulated after instigated inflammation of the Central Nervous System (CNS) and immune system activation. Here, we aimed to investigate the efficacy of PF‐06447475, an LRRK2 inhibitor, by counteracting pathological consequences of spinal cord trauma. The in vivo model of SCI was induced by extradural compression of the spinal cord at T6‐T8 levels, then mice were treated with PF‐06447475 (2.5‐5 and 10 mg/kg o.s) 1 and 6 hrs after SCI. We found that PF‐06447475 treatments at the higher doses (5 and 10 mg/kg) showed great abilities to reduce the degree of spinal cord tissue injury, glycogen accumulation, and demyelination of neurons associated with trauma. In addition, cytokines expression levels including interleukins (IL‐1, IL‐6, IL‐10 and12),interferong(IFN‐g)and tumor necrosis factor‐α (TNF‐α secreted and released by immune cells after trauma were decreased by LRRK‐2 inhibitor treatments. Moreover, the accumulation of CD4+and CD8+cells throughout the spinal cord lesion site of SCI mice was reduced by PF‐06447475 oral administration at the dose of 10 mg/kg. Taken together, our results suggest that exist a mutual correlation between LRRK2, neurodegeneration and immunity and that LRRK2 inhibition could have direct effects on the intervention of neuroinflammatory disorders.

Inhibition of LRRK2 Kinase Activity in the Treatment of Crohn’s Disease

Abstract The leucine-rich repeat kinase 2 (LRRK2) gene is a genetic hotspot for gain-of-function mutations associated with various diseases such as Crohn’s and Parkinson’s disease. These mutations enhance LRRK2 kinase activity and, consequently, various inhibitors of this activity are currently being tested as treatment modalities. Here, we report studies of two novel kinase inhibitors (Termed CS-190 and CS-82) with LRRK2 inhibition activity. In vitro studies disclosed that the two inhibitors suppress LRRK2 phosphorylation as well as its ability to phosphorylate the LRRK2 targets, Rab 10 and 12; in addition, they suppress human dendritic cell production of TNFa stimulated by several known LRRK2 activators including the Dectin-1-ligand, zymosan-depleted S. cerevisiae extract. Finally, the inhibitors exhibited powerful suppression of the ability of LRRK2 to mediate NLRC4 inflammasome production of IL-1beta. These in vitro finding correlated with in vivo studies showing that IP administration of one of the kinase inhibitors (CS-82) ameliorates colitis in the DSS-colitis model as evaluated by weight loss and inflammation scores. These findings suggest that these newly developed inhibitors are potential agents for treatment of Crohn’s disease.

Effect of LRRK2 protein and activity on stimulated cytokines in human monocytes and macrophages

Leucine-rich-repeat kinase 2 (LRRK2), a potential therapeutic target for the treatment of Parkinson’s disease (PD), is highly expressed in monocytes and macrophages and may play a role in the regulation of inflammatory pathways. To determine how LRRK2 protein levels and/or its activity modulate inflammatory cytokine/chemokine levels in human immune cells, isogenic human induced pluripotent stem cells (iPSC) with the LRRK2-activating G2019S mutation, wild-type LRRK2, and iPSC deficient in LRRK2 were differentiated to monocytes and macrophages and stimulated with inflammatory toll-like receptor (TLR) agonists in the presence and absence of LRRK2 kinase inhibitors. The effect of LRRK2 inhibitors and the effect of increasing LRRK2 levels with interferon gamma on TLR-stimulated cytokines were also assessed in primary peripheral blood-derived monocytes. Monocytes and macrophages with the LRRK2 G2019S mutation had significantly higher levels of cytokines and chemokines in tissue culture media following stimulation with TLR agonists compared to isogenic controls. Knockout of LRRK2 impaired phagocytosis but did not significantly affect TLR-mediated cytokine levels. Interferon gamma significantly increased the levels of LRRK2 and phosphorylation of its downstream Rab10 substrate, and potentiated TLR-mediated cytokine levels. LRRK2 kinase inhibitors did not have a major effect on TLR-stimulated cytokine levels. Results suggest that the LRRK2 G2019S mutation may potentiate inflammation following activation of TLRs. However, this was not dependent on LRRK2 kinase activity. Indeed, LRRK2 kinase inhibitors had little effect on TLR-mediated inflammation under the conditions employed in this study.

LRRK2 Inhibition Mitigates the Neuroinflammation Caused by TLR2-Specific α-Synuclein and Alleviates Neuroinflammation-Derived Dopaminergic Neuronal Loss.

Evidence suggests that crosstalk occurs between microglial leucine-rich repeat kinase 2 (LRRK2)—a regulator of neuroinflammation—and neuron-released α-synuclein (αSyn)—a promoter of microglial activation and neuroinflammatory responses—in neuroinflammation-mediated Parkinson’s disease (PD) progression. Therefore, we examined whether LRRK2 inhibition reduces the responses of microglia to neuroinflammation caused by neuron-released αSyn. We examined the neuroinflammatory responses provoked by Toll-like receptor 2 (TLR2)-positive αSyn of neuronal cells using an LRRK2 inhibitor in the mouse glioma cells, rat primary microglia, and human microglia cell line; and the effects of LRRK2 inhibitor in the co-culture of ectopic αSyn-expressing human neuroblastoma cells and human microglia cells and in mouse models by injecting αSyn. We analyzed the association between LRRK2 activity and αSyn oligomer and TLR2 levels in the substantia nigra tissues of human patients with idiopathic PD (iPD). The TLR2-specific αSyn elevated LRRK2 activity and neuroinflammation, and the LRRK2 inhibitor ameliorated neuroinflammatory responses in various microglia cells, alleviated neuronal degeneration along with neuroinflammation in the co-culture, and blocked the further progression of locomotor failure and dopaminergic neuronal degeneration caused by TLR2-specific αSyn in mice. Furthermore, LRRK2 phosphorylation was increased in patients with iPD showing αSyn-specific high TLR2 level. These results suggest the application of LRRK2 inhibitors as a novel therapeutic approach against αSyn-mediated PD progression.

Molecular Modeling Study on the Interaction Mechanism between the LRRK2 G2019S Mutant and Type I Inhibitors by Integrating Molecular Dynamics Simulation, Binding Free Energy Calculations, and Pharmacophore Modeling.

Leucine-rich repeat kinase 2 (LRRK2) has been reported in the pathogenesis of Parkinson's disease (PD). G2019S mutant is the most common pathogenic mutation in LRRK2-related PD patients. Inhibition of LRRK2 kinase activity is proposed to be a new therapeutic approach for PD treatment. Therefore, understanding the molecular basis of the interaction between LRRK2 and its inhibitors will be valuable for the discovery and design of LRRK2 inhibitors. However, the structure of human LRRK2 in complex with the inhibitor has not been determined, and the inhibitory mechanism underlying LRRK2 still needs to be further investigated. In this study, molecular dynamics (MD) simulation combined with the molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculation and pharmacophore modeling methods was employed to explore the critical residues in LRRK2 for binding of inhibitors and to investigate the general structural features of the inhibitors with diverse scaffolds. The results from MD simulations suggest that the hinge region residues Glu1948 and Ala1950 play a significant role in maintaining the intermolecular hydrogen bond interaction with the G2019S LRRK2 protein and inhibitor. The strong hinge hydrogen bond with an occupancy rate of more than 95% represents the high activity of LRRK2 inhibitors, and the hydrogen bond interaction with the kinase catalytic loop region could compromise selectivity. Further pharmacophore modeling reveals that the high activity LRRK2 inhibitor should have one aromatic ring, one hydrogen bond acceptor, and one hydrogen bond donor. Hence, the obtained results can provide valuable information to understand the interactions of LRRK2 inhibitors at the atomic level that will be helpful in designing potent inhibitors of LRRK2.

Exploring the focal role of LRRK2 kinase in Parkinson's disease.

The major breakthroughs in our knowledge of how biology plays a role in Parkinson's disease (PD) have opened up fresh avenues designed to know the pathogenesis of disease and identify possible therapeutic targets. Mitochondrial abnormal functioning is a key cellular feature in the pathogenesis of PD. An enzyme, leucine-rich repeat kinase 2 (LRRK2), involved in both the idiopathic and familial PD risk, is a therapeutic target. LRRK2 has a link to the endolysosomal activity. Enhanced activity of the LRRK2 kinase, endolysosomal abnormalities and aggregation of autophagic vesicles with imperfectly depleted substrates, such as α-synuclein, are all seen in the substantia nigra dopaminergic neurons in PD. Despite the fact that LRRK2 is involved in endolysosomal and autophagic activity, it is undefined if inhibiting LRRK2 kinase activity will prevent endolysosomal dysfunction or minimise the degeneration of dopaminergic neurons. The inhibitor's capability of LRRK2 kinase to inhibit endolysosomal and neuropathological alterations in human PD indicates that LRRK2 inhibitors could have significant therapeutic usefulness in PD. G2019S is perhaps the maximum common mutation in PD subjects. Even though LRRK2's well-defined structure has still not been established, numerous LRRK2 inhibitors have been discovered. This review summarises the role of LRRK2 kinase in Parkinson's disease.

Inhibition of LRRK2-Rab10 Pathway Improves Secondary Brain Injury After Surgical Brain Injury in Rats.

Leucine-rich repeat kinase 2 (LRRK2) is considered as a potential target for the treatment of Parkinson's disease. This protein is expressed in the brain and has been associated with various diseases and lysosomal maintenance. Rab10 is a member of the Rab protein GTPase family that has been recently shown to be a kinase substrate of LRRK2. In addition, LRRK2 and its kinase substrate Rab10 constitute a key stress response pathway during lysosomal overload stress. This study aimed to investigate the potential role and mechanism underlying LRRK2 and its kinase substrate Rab10 involving surgical brain injury (SBI). One hundred and forty-four male Sprague-Dawley rats were examined using an SBI model, and some had received the LRRK2-specific inhibitor PF-06447475. Thereafter, western blotting, immunofluorescence, brain water content analysis, neuronal apoptosis assay, and neurological score analysis were conducted. The results showed that after SBI, LRRK2 and phosphorylated Rab10 (p-Rab10) expression in neuronal cells were upregulated, and administration of PF-06447475 significantly reduced neuronal apoptosis, neuroinflammation, and brain water content 12 h after SBI and improved neurological deficit 72 h after SBI, which is related to the decreased expression of LRRK2 and p-Rab10, and the lessening of lysosomal overload stress. Our research suggests that the inhibition of LRRK2 can effectively interfere with the role of p-Rab10 in promoting the secretion of lysosomal hydrolase in lysosomal overload stress after SBI, thereby reducing neuronal apoptosis and inflammation after SBI and playing a major role in brain protection.

Structure-Guided Discovery of Aminoquinazolines as Brain-Penetrant and Selective LRRK2 Inhibitors.

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24. These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.

Inhibition of LRRK2 kinase activity promotes anterograde axonal transport and presynaptic targeting of \u03b1-synuclein

Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson’s Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

Allosteric Inhibition of Parkinson's-Linked LRRK2 by Constrained Peptides.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson's Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

Virtual Screening and Biological Activity Evaluation of New Potent Inhibitors Targeting LRRK2 Kinase Domain

Leucine rich repeat kinase 2 (LRRK2) has been reported in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may provide new treatments for PD. In this study, novel LRRK2 inhibitors were identified by performing a docking-based virtual screening (VS). Due to the absence of a crystal structure of LRRK2, homology modeling was adopted to model human LRRK2 kinase domain that binds the inhibitor. Next, a docking-based virtual screening protocol was applied to identify LRRK2 small molecule inhibitors targeting the ATP binding pocket. A total of 28 compounds were selected and subjected to LRRK2 kinase inhibition assay. As a result, two small molecules with novel skeleton, compounds LY2019-005 and LY2019-006, were identified as potential LRRK2 kinase inhibitors with the IC50 of these two compounds against the wild-type and G2019S mutant LRRK2 kinase being 424.40 ± 1.31 nM, 378.80 ± 1.20 nM and 1526.00 ± 0.87 nM, 1165.00 ± 1.18 nM, respectively. Molecular dynamics (MD) simulation was carried out to reveal the binding mode of the newly identified compound LY2019-005 to the LRRK2 kinase domain. The binding modes indicate that the important hydrogen bond between hinge region (such as Ala1950) and inhibitor is crucial for the inhibition activity. In summary, our study provides a highly efficient way to discover LRRK2 inhibitors, and we find two highly efficient novel LRRK2 inhibitors, which could be helpful for the development of potential drugs targeting LRRK2 in PD therapy.

LRRK2 Targeting Strategies as Potential Treatment of Parkinson's Disease.

Parkinson’s Disease (PD) affects millions of people worldwide with no cure to halt the progress of the disease. Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of PD and, as such, LRRK2 inhibitors are promising therapeutic agents. In the last decade, great progress in the LRRK2 field has been made. This review provides a comprehensive overview of the current state of the art, presenting recent developments and challenges in developing LRRK2 inhibitors, and discussing extensively the potential targeting strategies from the protein perspective. As currently there are three LRRK2-targeting agents in clinical trials, more developments are predicted in the upcoming years.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of familial Parkinson’s disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S+/- mice. However, a significant increase in ciliogenesis was observed in G2019S+/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

Design and Synthesis of Pyrrolo[2,3-d]pyrimidine-Derived Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Checkpoint Kinase 1 (CHK1)-Derived Crystallographic Surrogate.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

The development of inhibitors of leucine-rich repeat kinase 2 (LRRK2) as a therapeutic strategy for Parkinson's disease: the current state of play.

Current therapeutic approaches for Parkinson's disease (PD) are based around treatments that alleviate symptoms but do not slow or prevent disease progression. As such, alternative strategies are needed. A promising approach is the use of molecules that reduce the function of leucine-rich repeat kinase (LRRK2). Gain-of-function mutations in LRRK2 account for a notable proportion of familial Parkinson's disease cases, and significantly, elevated LRRK2 kinase activity is reported in idiopathic Parkinson's disease. Here, we describe progress in finding therapeutically effective LRRK2 inhibitors, summarising studies that range from in vitro experiments to clinical trials. LRRK2 is a complex protein with two enzymatic activities and a myriad of functions. This creates opportunities for a rich variety of strategies and also increases the risk of unintended consequences. We comment on the strength and limitations of the different approaches and conclude that with two molecules under clinical trial and a diversity of alternative options in the pipeline, there is cause for optimism. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.