Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation

Hertie Institute for Clinical Brain Research and German Center for Neurodegenerative Diseases, Germany
PLoS ONE (Impact Factor: 3.23). 08/2012; 7(8):e43580. DOI: 10.1371/journal.pone.0043580
Source: PubMed


Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.
Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.
We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

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Available from: R. Jeremy Nichols, Aug 21, 2014
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    • "We found that all the cellular phosphorylation sites were significantly diminished by approximately 20% in a PKA activated environment and pSer1292 was not significantly changed. These data are in agreement with a recent report by Hermanson et al. (2012), which analyzed pSer935 levels following treatments with various LRRK2 inhibitors. In that report, PKA inhibitor H89 was not effective or had high cellular IC50 values compared other LRRK2 inhibitors in unstimulated HEK293 and U20S and SHSY5Y cells. "
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    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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    • "The same holds true for the second assay testing cellular activity which is based on kinase inhibitor induced dephosphorylation of LRRK2 at Ser935 and adapted here using a spotblot readout. For instance, for LRRK2-IN1 the IC50 value obtained here (279.9 nM) is in the same range as IC50 values obtained with other readouts of pS935 levels such as western blot (about 100 nM; Dzamko et al., 2010; Deng et al., 2011), time-resolved FRET (90–200 nM; Hermanson et al., 2012), or ELISA (50–100 nM; Delbroek et al., 2013). Thirdly, in the absence of a physical 3D atomic structure of the LRRK2 kinase domain, an optimized 3D homology model was generated for in silico docking. "
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    ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson’s disease. In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modelled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells results from compound activity on the LRRK2 ATP-binding pocket itself. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.
    Frontiers in Molecular Neuroscience 05/2014; 7:51. DOI:10.3389/fnmol.2014.00051 · 4.08 Impact Factor
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    ABSTRACT: Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.
    Biochemical Society Transactions 10/2012; 40(5):1158-62. DOI:10.1042/BST20120137 · 3.19 Impact Factor
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