Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease
ABSTRACT The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.
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- "Several potent, non-selective tool compounds, including staurosporine (LRRK2: IC 50 ¼ 1e2 nM) , sunitinib (LRRK2: IC 50 ¼ 79 nM)  and ROCK inhibitor H-1152 (LRRK2: IC 50 ¼ 244 nM)  provided preliminary support for LRRK2 inhibition as a potential therapeutic option for PD. Subsequent studies focused on developing inhibitors selective for LRRK2. "
ABSTRACT: LRRK2IN1 is a highly potent inhibitor of leucine-rich repeat kinase 2 (LRRK2, IC50 = 7.9 nM), an established target for treatment of Parkinson's disease. Two LRRK2IN1 analogues 1 and 2 were synthesised which retained LRRK2 inhibitory activity (1: IC50 = 72 nM; 2: IC50 = 51 nM), were predicted to have improved bioavailability and were efficacious in cell-based models of neuroinflammation. Analogue 1 inhibited IL-6 secretion from LPS-stimulated primary human microglia with EC50 = 4.26 μM. In order to further optimize the molecular properties of LRRK2IN1, a library of truncated analogues was designed based on docking studies. Despite lacking LRRK2 inhibitory activity, these compounds show anti-neuroinflammatory efficacy at micromolar concentration. The compounds developed were valuable tools in establishing a cell-based assay for assessing anti-neuroinflammatory efficacy of LRRK2 inhibitors. Herein, we present data that IL-1β stimulated U87 glioma cell line is a reliable model for neuroinflammation, as data obtained in this model were consistent with results obtained using primary human microglia and astrocytes. Copyright © 2015 Elsevier Masson SAS. All rights reserved.European Journal of Medicinal Chemistry 05/2015; 95. DOI:10.1016/j.ejmech.2015.03.003 · 3.43 Impact Factor
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- "Threonines 1491 and 2483 were evaluated in the presence of ATP and we found that Thr1491 revealed significant increase in modification from the Gly2019Ser mutation, while Thr2483 revealed significant increased kinase activity from Arg1441Gly, and Gly2019Ser. Both Ile2020Thr and Ile2012Thr mutations reduced the basal level of Ser1292 and autophosphorylation activity similar to activity determinations using LRRKtide and Nictide (Jaleel et al., 2007; Nichols et al., 2009). The Ala1442Pro mutant revealed reduced levels of phosphorylation at Thr1491 and Ser1292. "
ABSTRACT: Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.Frontiers in Molecular Neuroscience 06/2014; 7:54. DOI:10.3389/fnmol.2014.00054 · 4.08 Impact Factor
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- "Using an automated version of the digital morphological assay described earlier, we tested the effect of several commercially available ROCK inhibitors (ROCKi; Figure 2 and Table 1) on the differentiation of mouse Nph OPC. These inhibitors included Fasudil, a ROCK inhibitor in use in Japan for more than 15 years for the treatment of brain vasospasm (Hirooka & Shimokawa, 2005), and two other small molecules widely used in ROCK basic research in diverse areas: Y-27632 (trans-4-[(1 R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride; Koyanagi et al., 2008; Narumiya et al., 2000) and GSK 429286: 4-[4-(trifluoromethyl)phenyl]-N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide; Goodman et al., 2007; Nichols et al., 2009). Generation of branches was measured in dose-response experiments yielding IC50 values of ∼2.5 to 2.9 µM for these compounds (Figure 2(a)). "
ABSTRACT: In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.ASN Neuro 06/2014; 6(4). DOI:10.1177/1759091414538134 · 4.44 Impact Factor