MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition

Fell, Matthew; Mirescu, Christian; Basu, Kallol; Cheewatrakoolpong, Boonlert; DeMong, Duane E.; Ellis, J. Michael; Hyde, Lynn A.; Lin, Yuan; Markgraf, Carrie G.; Hong, Mei; Miller, Mark W.; Poulet, Frederique M.; Scott, Jack D.; Smith, Michelle; Yin, Zhizhang; Zhou, Xiaoping; Parker, Eric M.; Kennedy, Matthew; Morrow, John A.

doi:10.1124/jpet.115.227587

Cited by 251 publications

(331 citation statements)

References 32 publications

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“…LRRK2 phosphorylation induced a time-dependent retardation in the migration of phosphorylated Rab10, an effect that was prevented by inclusion of the MLi-2 LRRK2 kinase inhibitor in the kinase reaction [36] (Figure 1A). Immunoblot analysis with a phospho-specific antibody confirmed that the slower migrating Rab10 species that appears following LRRK2 phosphorylation is indeed Rab10 phosphorylated at Thr 73 (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Ito

Katsemonova

Tonelli

et al. 2016

Biochemical Journal

149

191

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Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

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Section: Resultsmentioning

confidence: 99%

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Ito

Katsemonova

Tonelli

et al. 2016

Biochemical Journal

149

191

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“…Likewise, LRRK2 knockouts are not protected from the mitochondrial complex I inhibitor MPP that is selectively internalized in DAT-1 expressing neurons(Andres-Mateos et al, 2009). Protection was also not recorded in mice treated with a LRRK2 kinase inhibitor in the MitoPark mice that have neurodegeneration caused by mitochondrial deficits in DAT-1 expressing neurons(Fell et al, 2015). Thus, from these studies it can be deduced that neuroprotection associated with LRRK2 inhibition may be due to reductions in neuroinflammation, potentially related to the damaging effects of wild-type α-synuclein over-expression.…”

Section: Evidence For Lrrk2 Kinase Inhibition In Neuroprotectionmentioning

confidence: 99%

“…Chronic oral dosing of both rats and mice with third-generation LRRK2 kinase inhibitors has been described for the MLi-2 compound, as well as for the PFE475 compound(Daher et al, 2015; Fell et al, 2015). In these studies, dosing that achieved near-continuous brain LRRK2 inhibition (up to 90% inhibition for much of the day) did not produce clear phenotypes associated with the LRRK2 knockout in rodents, and only mild or non-significant effects on reducing total LRRK2 protein.…”

Section: Safety Of Lrrk2 Inhibitionmentioning

confidence: 99%

“…Fortunately, the available data supports the notion of a reasonable therapeutic window of opportunity. In rats and mice treated chronically (days and weeks) with doses of inhibitors that do not fully block LRRK2 kinase activity (e.g., 30 mg per kg daily or twice daily with MLi-2 or PFE360 and PFE475), total LRRK2 levels are only slightly reduced(Daher et al, 2015; Fell et al, 2015). NHPs treated with lower levels of inhibitors that still block nearly all the LRRK2 kinase activity did not cause lung abnormalities(Baptista et al, 2015; Fuji et al, 2015).…”

Section: Biomarkers For Lrrk2 Inhibition and Therapeutic Windows Fmentioning

confidence: 99%

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Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease

West

2017

Experimental Neurology

134

140

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In the translation of discoveries from the laboratory to the clinic, the track record in developing disease-modifying therapies in neurodegenerative disease is poor. A carefully designed development pipeline built from discoveries in both pre-clinical models and patient populations is necessary to optimize the chances for success. Genetic variation in the leucine-rich repeat kinase two gene (LRRK2) is linked to Parkinson disease (PD) susceptibility. Pathogenic mutations, particularly those in the LRRK2 GTPase (Roc) and COR domains, increase LRRK2 kinase activities in cells and tissues. In some PD models, small molecule LRRK2 kinase inhibitors that block these activities also provide neuroprotection. Herein, the genetic and biochemical evidence that supports the involvement of LRRK2 kinase activity in PD susceptibility is reviewed. Issues related to the definition of a therapeutic window for LRRK2 inhibition and the safety of chronic dosing are discussed. Finally, recommendations are given for a biomarker-guided initial entry of LRRK2 kinase inhibitors in PD patients. Four key areas must be considered for achieving neuroprotection with LRRK2 kinase inhibitors in PD: 1) identification of patient populations most likely to benefit from LRRK2 kinase inhibitors , 2) prioritization of superior LRRK2 small molecule inhibitors based on open disclosures of drug performance, 3) incorporation of biomarkers and empirical measures of LRRK2 kinase inhibition in clinical trials, and 4) utilization of appropriate efficacy measures guided in part by rigorous pre-clinical modeling. Meticulous and rational development decisions can potentially prevent incredibly costly errors and provide the best chances for LRRK2 inhibitors to slow the progression of PD.

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“…Its ability to inhibit LRRK2 in vivo reveals that it can cross the BBB. Since its description in 2012, several novel, selective, and brain-penetrating LRRK2 inhibitors have been described, including GSK2578215A [60], GNE-7915 [61], JH-II-127 [62], PF-06447475 [63], and MLi-2 (Table 1 for further details) [64]. These next-generation LRRK2 inhibitors exhibit improved potency, selectivity, and brain penetrability, all of which are important for clinical application.…”

Section: Lrrk2 As a Therapeutic Targetmentioning

confidence: 99%

Leucine-Rich Repeat Kinase 2 in Parkinson’s Disease: Updated from Pathogenesis to Potential Therapeutic Target

Chen¹,

Chen²,

Pu³

2018

Eur Neurol

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Background: Parkinson’s disease (PD) is characterized by the selective loss of dopaminergic neurons in the midbrain. The pathogenesis of PD is not fully understood but is likely caused by a combination of genetic and environmental factors. Several genes are associated with the onset and progression of familial PD. There is increasing evidence that leucine-rich repeat kinase 2 (LRRK2) plays a significant role in PD pathophysiology. Summary: Many studies have been conducted to elucidate the functions of LRRK2 and identify effective LRRK2 inhibitors for PD treatment. In this review, we discuss the role of LRRK2 in PD and recent progress in the use of LRRK2 inhibitors as therapeutic agents. Key Messages: LRRK2 plays a significant role in the pathophysiology of PD, and pharmacological inhibition of LRRK2 has become one of the most promising potential therapies for PD. Further research is warranted to determine the functions of LRRK2 and expand the applications of LRRK2 inhibitors in PD treatment.

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MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition

Cited by 251 publications

References 32 publications

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease

Leucine-Rich Repeat Kinase 2 in Parkinson’s Disease: Updated from Pathogenesis to Potential Therapeutic Target

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