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.     

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     

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 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.     

Synthesis of [11C]HG-10-102-01 as a new potential PET agent for imaging of LRRK2 enzyme in Parkinson’s disease

The reference standard (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(morpholino)methanone (HG-10-102-01) and its precursor (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-hydroxyphenyl)(morpholino)methanone (desmethyl-HG-10-102-01) were synthesized from 2,4,5-trichloropyrimide and 3-methoxy-4-nitrobenzoic acid with overall chemical yield 49% in four steps and 14% in five steps, respectively. The target tracer (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-[¹¹C]methoxyphenyl)(morpholino)methanone ([¹¹C]HG-10-102-01) was prepared from the precursor desmethyl-HG-10-102-01 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 45–55% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB.     

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.     

A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain enzyme displaying activities of GTP hydrolase and protein threonine/serine kinase in separate domains. Mutations in both catalytic domains have been linked to the onset of Parkinson’s disease, which triggered high interest in this enzyme as a potential target for drug development, particularly focusing on inhibition of the kinase activity. However, available activity assays are discontinuous, involving either radioactivity detection or coupling with antibodies. Here we describe a continuous and direct assay for LRRK2 kinase activity, combining a reported peptide sequence optimized for LRRK2 binding and an established strategy for fluorescence emission on magnesium ion chelation by phosphorylated peptides carrying an artificial amino acid. The assay was employed to evaluate apparent steady-state parameters for the wild type and two mutant forms of LRRK2 associated with Parkinson’s disease as well as to probe the effects of GTP, GDP, and autophosphorylation on the kinase activity of the enzyme. Staurosporine was evaluated as an inhibitor of the wild-type enzyme. It is expected that this assay will aid in mechanistic investigations of LRRK2.     

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.     

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.     

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.     

Structural analysis of the full-length human LRRK2

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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.     

帕金森病相关蛋白质LRRK2在脑中的表达分布与定位分析

帕金森病（Parkinson’s disease,PD）是一种最常见的神经退行性运动障碍,常染色体显性遗传PD可由LRRK2 (leucine-rich repeat kinase 2)基因突变引起.原核表达和纯化得到LRRK2多肽与GST的融合蛋白,免疫新西兰雄兔,得到了anti-LRRK2兔多克隆抗体. 抗体用途分析实验表明,自制的anti-LRRK2兔多克隆抗体可用于Western 印迹, 免疫组织化学,免疫细胞染色等实验.利用自制的抗体,Western 印迹证明,LRRK2在大鼠脑主要神经解剖学部位均有表达.免疫荧光双染色的结果表明,LRRK2定位于线粒体.本研究对了解LRRK2在PD中的分子生物学功能具有重要的意义.     

LRRK2 regulates synaptic vesicle endocytosis

The leucine-rich repeat kinase 2 (LRRK2) has been identified as the defective gene at the PARK8 locus causing the autosomal dominant form of Parkinson's disease (PD). Although several LRRK2 mutations were found in familial as well as sporadic PD patients, its physiological functions are not clearly defined. In this study, using yeast two-hybrid screening, we report the identification of Rab5b as an LRRK2-interacting protein. Indeed, our GST pull down and co-immunoprecipitation assays showed that it specifically interacts with LRRK2. In addition, subcellular fractionation and immunocytochemical analyses confirmed that a fraction of both proteins co-localize in synaptic vesicles. Interestingly, we found that alteration of LRRK2 expression by either overexpression or knockdown of endogenous LRRK2 in primary neuronal cells significantly impairs synaptic vesicle endocytosis. Furthermore, this endocytosis defect was rescued by co-expression of functional Rab5b protein, but not by its inactive form. Taken together, we propose that LRRK2, in conjunction with its interaction with Rab5b, plays an important role in synaptic function by modulating the endocytosis of synaptic vesicles.     

Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson's disease R1441C/G mutants

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson's disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD.     

Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO^LRRK2 self-interaction. Interestingly, ROCO^LRRK2 fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO^LRRK2 reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO^LRRK2 fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.     

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.     

A Neuron-Glial Trans-Signaling Cascade Mediates LRRK2-Induced Neurodegeneration

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