Spencer Hermanson, Coby Carlson, Steve Riddle, Kurt Vogal and Kun Bi are employees of Life Technologies, which manufactures some of the products/tools used in this study. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
Conceived and designed the experiments: SH RJN KB. Performed the experiments: SH CC SR JZ RJN KB. Analyzed the data: SH CC SR JZ RJN KB. Contributed reagents/materials/analysis tools: SH CC SR JZ KV RJN KB. Wrote the paper: RJN KB.
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
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60 and more than 5 million people worldwide. PD results primarily from the selective loss of dopaminergic neurons in the substantia nigra. Current available therapies only address the symptoms of PD, and the most effective treatment has been in use for more than 50 years. The identification of familial mutations associated with PD has presented novel targets for potential therapeutic development. Mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal familial and sporadic PD
LRRK2 is a large multi-domain protein that is phylogenetically related to the Receptor Interacting Kinase branch of the tyrosine kinase-like family of the human kinome. Indeed the kinase domain does bear homology to MAP3K kinases. It contains several potential protein-protein interaction domains including N-terminal ankyrin repeats, leucine-rich repeats and a C-terminal WD40 domain. Surrounded by these domains is the catalytic core containing a GTP-binding Ras of complex protein (ROC) GTPase domain, a carboxy-terminal of Roc (COR) domain and a serine/threonine kinase domain
LRRK2 is found to be phosphorylated at multiple sites throughout the protein and can be classified as autophosphorylation sites or sites of constitutive phosphorylation, which are detected on active LRRK2 expressed in cells. Multiple autophosphorylation sites on LRRK2 have been mapped
Several biochemical assay formats have been reported and utilized for measuring
The first step for developing a LanthaScreen® TR-FRET cellular assay is to generate cells expressing substrate of interest fused to GFP which acts as the fluorescence acceptor from a terbium labeled modification-specific antibody
U-2 OS cells were transduced with 20% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. (A) GFP images were captured and representative images are shown. (B) Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody. The quantifications are from 3 independent experiments.
The second step for developing a LanthaScreen® TR-FRET cellular assay is to generate terbium labeled modification-specific antibodies. Here, the same antibody that was used in the Western blot analysis for Ser935 phosphorylation (
(A) U-2 OS cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were plated onto a 384-well assay plate and lysed in the presence of Tb-labeled anti-pSer935 antibody. TR-FRET was measured and the emission ratios of 520 nm/490 nm are plotted against the amount of BacMam used for transduction. (B) Cells transduced with 20% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (A). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.
(A) SH-SY5Y cells were transduced with 25% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody was performed. The quantifications are from 3 independent experiments. (B) SH-SY5Y cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C and D1994A. Cells were plated onto a 384-well assay plate, lysed and analyzed as described in
Human neural stem cells were transduced with 10% BacMam LRRK2-GFP G2019S or D1994A. Cells were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in
To further validate the TR-FRET cellular assay, LRRK2-IN-1 and seven other small molecule inhibitors previously reported to have inhibitory activities of LRRK2 kinase were profiled in three different cell backgrounds, U-2 OS, SH-SY5Y and HEK293T transduced with BacMam LRRK2-GFP wild-type or G2019S. The IC50 values of these inhibitors are summarized in
U-2 OS | SH-SY5Y | HEK293T | ||||
Compound | WT | G2019S | WT | G2019S | WT | G2019S |
LRRK2-IN-I | 0.09+/−0.05 | 0.05+/−0.02 | 0.20+/−0.03 | 0.06+/−0.03 | 0.12+/−0.04 | 0.06+/−0.03 |
TAE684 | 0.09+/−0.04 | 0.07+/−0.01 | 0.06+/−0.02 | 0.03+/−0.01 | 0.12+/−0.04 | 0.10+/−0.03 |
JAK3 Inh. VI | 0.57+/−0.19 | 0.17+/−0.04 | 2.77+/−0.76 | 0.68+/−0.07 | 0.81+/−0.31 | 0.26+/−0.05 |
Indirubin-3-monoxime | 3.78+/−0.10* | 1.93+/−0.09* | >10 | 6.15+/−2.75* | 3.68+/−2.03* | 2.78+/−1.10* |
Sunitinib | 0.29+/−0.07 | 0.16+/−0.01 | 0.62+/−0.36 | 0.32+/−0.07 | 0.45+/−0.14 | 0.32+/−0.16 |
GW5074 | >20 | 0.47+/−0.14* | >20 | >20 | >20 | 0.28+/−0.12* |
H-1152 | 3.28+/−0.69 | 0.89+/−0.05 | 1.66+/−0.04 | 0.77+/−0.20 | 2.94+/−0.98 | 1.38+/−0.58 |
H-89 | >20 | >20 | >20 | >20 | nd | nd |
IC50 (µM) +/− SD; *Partial inhibition; nd: not tested.
To test the performance of this assay in a high-throughput screen, Tocriscreen™ Mini library of 1120 biologically active compounds which target ion channels, G-protein-coupled receptors (GPCRs), nuclear receptors and kinases were screened in the TR-FRET Ser935 phosphorylation cellular assay using BacMam LRRK2-GFP G2019S transduced SH-SY5Y cells. Each compound was tested twice at a single concentration of 20 µM in 0.1% DMSO using duplicate 384-well assay plates, to reproduce actual conditions that may be used in a larger high-throughput screen. LRRK2-IN-1 was included as a positive control on each plate and used at 10 µM. Percent inhibition of each compound is calculated and plotted in a histogram shown in
Average percent inhibition of library compounds was analyzed to produce a frequency distribution with binning equal to one percent increments. The frequency distribution was plotted and a gaussian curve fit to the distribution (curve statistics: mean 4.38% standard, deviation 10.53% and R2 value of 0.962).
Out of the 1120 compounds, 16 compounds reduced the level of Ser935 phosphorylation by greater than 50%. Among the 16 compounds, SP600125, indirubin-3′-monoxime and ROCK inhibitor Y-27632 with previously reported LRRK2 inhibitory activity showed 88%, 67% and 63% inhibition, respectively in this screen (
%Inhibition | Compound | Known Activity | Cellular TR-FRET Assay | Cytotoxicity Assay | Biochemical LRRK2 assay | |
Initial screen | Max % Inhibition | IC50 (µM) +/−SD | IC50 (µM) | IC50 (µM) +/− SD | ||
97.5 | Bay 11-7085 | Irreversible inhibitor of TNFa-induced IkB phosphorylation | 100 | 5.3+/−2.8 | >20 | >100 |
90.1 | JTC 801 | ORL1 antagonist | 84 | 9.6+/−6.3 | 10 | >100 |
88.3 | SP 600125 | JNK and other kinase inhibitor | 103 | 1.0+/−0.4 | >20 | 0.22+/−0.16 |
85.5 | Bay 11-7821 | Irreversible inhibitor of TNFa-induced IkB phosphorylation | 94 | 4.0+/−2.1 | 20 | >100 |
81.7 | IKK 16 | Inhibitor of IKK | 82 | 1.3+/−0.8 | >20 | 0.05+/−0.03 |
76.1 | NSC 95397 | Selective Cdc25 dual specificity phosphatase inhibitor | 80 | 1.3+/−0.9 | 7.8 | 15+/−5.5 |
69.8 | Ro 106-9920 | Inhibitor of NFkB activation | 76 | 7.6+/−0.01 | 20 | >100 |
69.7 | GW 441756 | TrkA inhibitor | 72 | 2.2+/−0.3 | >20 | 0.32+/−0.18 |
69.7 | BNTX maleate | Standard d1 selective antagonist | 88 | 4.0+/−1.4 | >20 | >100 |
67.3 | PD 407824 | Inhibitor of Chk1 and Wee1 | 65 | 0.5+/−0.04 | 15 | 0.32+/−0.32 |
67.3 | Y-27632 | p160ROCK inhibitor | 69 | 10+/−4.7 | >20 | 0.57+/−0.27 |
63.5 | Indirubin-3-oxime | GSK-3b inhibitor. Also inhibits other protein kinases | 64 | 4.4+/−0.5 | >20 | >10 |
59.1 | NSC 663284 | Cdc25 phosphatase inhibitor | 45 | 5.8+/−1.2 | 2 | 0.48+/−0.32 |
56.2 | Aminopurvalanol A | Cyclin-dependent kinase inhibitor | 50 | 1.4+/−2.2 | >20 | ND |
55.3 | SB 218078 | Inhibitor of checkpoint kinase 1 (Chk1) | 54 | 9.5+/−0.4 | >20 | ND |
54.8 | TPCA-1 | Inhibitor of IKK-2 | 50 | 0.1+/−0.03 | >20 | ND |
100 | LRRK2 IN-1 | Positive Control Compound | 100 | 0.07+/−0.02 | >20 | 0.01+/−0.001 |
The IC50 values are the averaged numbers from three independent experiments for both cellular and biochemical TR-FRET assays and one representative data of two independent experiments for the cytotoxicity assay.
From these initial screens, we evaluated 13 of these compounds further by determining the
To further confirm the TR-FRET cellular assay results, we also evaluated the effects of these compounds on the cellular phosphorylation of Ser910, Ser935, Ser955 and Ser973 by immunoprecipitation (IP) followed by Western analysis. Here, commonly used HEK293 cells inducibly expressing N-terminal GFP tagged LRRK2 G2019S
Flp-In T-REx™ HEK293 GFP-LRRK2 G2019S cells (GS) or A2016T/G2019S (IRM) were induced and treated with indicated compounds at 20 µM. After cell lysis, GFP-LRRK2 was immunoprecipitated and the phosphorylation of LRRK2 was analyzed by immunoblotting. The quantifications are from 3 independent experiments.
We further analyzed the compounds that were found to directly inhibit LRRK2 assays, using the T-REx cellular system expressing an inhibitor desensitizing mutant LRRK2 A2016T/G2019S
We have developed a high-throughput and homogenous cellular TR-FRET immunoassay for the measurement of LRRK2 phosphorylation. Since acute inhibition of LRRK2 kinase activity can reduce the level of Ser935 phosphorylation, this assay can be applied to high-throughput cell-based screens for LRRK2 kinase inhibitors. Screening of a small molecule inhibitor library with this assay indeed revealed several inhibitors with previously unknown LRRK2 activity as well as provided leads to cellular pathways that could involve LRRK2.
This high-throughput assay utilizes cells expressing full-length human LRRK2 with a C-terminal GFP tag (LRRK2-GFP). We provide multiple lines of evidence suggesting that LRRK2-GFP functions and behaves similarly to the previously reported N-terminally tagged LRRK2 (GFP-LRRK2) stably expressed in HEK293 cells. First, wild-type and G2019S LRRK2-GFP displayed a diffuse cytoplasmic localization
Second, LRRK2-GFP wild-type, G2019S, R1441C and D1994A showed similar Ser935 phosphorylation pattern determined by Western blot and the TR-FRET assay as reported for GFP-LRRK2
One of the common methods for detecting LRRK2 phosphorylation is immunoblotting, typically after immunoprecipation. The complexities of this methodology make it highly impractical for the processing and analysis of large sample numbers typically associated with screening experiments. The TR-FRET cellular assay reported here is in a fully homogenous format without washing, lysate transfer, or separation procedures. Cells transduced with BacMam LRRK2-GFP can be plated onto a 384-well assay plate, treated as desired and then incubated with 6 X lysis buffer containing Tb-labeled detection antibody added directly to the wells. To further simplify the procedure, we demonstrated that cells can be transduced, cryopreserved, later thawed and used directly to run the assay without the need for culturing (
We profiled 1120 compounds of the Tocriscreen™ Mini library to gain insight into the biology of Ser935 phosphorylation and demonstrate the utility of the TR-FRET cellular assay in screening. Using a maximal inhibition threshold of 50%, we observed the previously identified inhibitors of LRRK2 SP600125, indirubin-3′-monoxime and ROCK inhibitor Y-27632 induced dephosphorylation of Ser935, which increased confidence in our assays. In addition to compounds with known LRRK2 inhibitory activity, we identified several novel LRRK2 kinase inhibitors from this screen. GW441756 is a known TrkA inhibitor
Interestingly, we observed that 5 out of the top 16 compounds Bay 11-7085, Bay 11-7821, IKK16, Ro106-9920 and TPCA-1 intersect with the NFκB pathway. TPCA-1 and IKK16 are IKK inhibitors, however LRRK2 is inhibited by IKK16 at an
Inhibitors of the Cdc25 phosphatase NSC-95397 and NSC-663284 or kinase inhibitors aminopurvalanol A (CDK), SB 218078 and PD407824 (CHK1) can block cell cycle progression
We also observed compounds other than kinase inhibitors that affect LRRK2 phosphorylation, but do not directly target LRRK2 enzyme itself, namely GPCR ligands such as opioid-receptor like-1 (OPRL-1) antagonist JTC801, the dopamine receptor D1 antagonist BNTX maleate, dopamine D2-like receptor ligand 3′-Fluorobenzylspiperone maleate, and an agonist of the adenosine A2 receptor CV1808. Though results are complicated for some of these compounds due to their cytotoxic effects, these compounds could indicate potential signaling pathways that also alter LRRK2 function via effects on LRRK2 phosphorylation. These represent avenues for further research.
In summary, we have developed a robust high-throughput detection system for evaluating the phosphorylation of LRRK2 at Ser935 that correlates well with alternate systems. A small library was used to evaluate this screening system and hits from that screen were validated in established systems showing the reliability of the methodology. By exploiting the biology of Ser935 phosphorylation, we gained novel insights into potential pathways that intersect with LRRK2, as well as identified other possible direct LRRK2 inhibitors.
ReagentsAll cell culture media and supplements, BacMam LRRK2-GFP reagents, PrestoBlue™ cell viability assay reagent, Western blotting reagents and human neural stem cells were obtained from Life Technologies (Carlsbad, CA). All other cell lines were purchased from ATCC (Manassas, VA). The cell lines were maintained under conditions recommended by the manufacturer. Anti-LRRK2 antibody used for Western blotting was purchased from Cell Signaling Technologies (Danvers, MA). Protease and phosphatase inhibitor cocktails and H-89 were purchased from Sigma (St. Louis, MO). LRRK2-IN-1 was provided by the Michael J. Fox Foundation for Parkinson’s Research. Sunitinib was from LC Laboratories (Woburn, MA), JAK3 Inhibitor VI and Indirubin-3′-monoxime were from EMD Biosciences (San Diego, CA). H-1152, GW5074 and the Tocriscreen™ Mini library and the follow-up compounds were from Tocris (Ellisville, MO). Rabbit polyclonal anti-phospho Ser910, Ser935, Ser955 and Ser973 antibodies were produced by Yenzym Antibodies (South San Francisco, CA) as described in
U-2 OS cells are maintained in growth medium (McCoy’s 5A supplemented with 10% dialyzed FBS (dFBS), 10 mM HEPES, 0.1 mM NEAA, 1 mM Sodium Pyruvate, and 100 U/mL Penicillin/100 µg/mL Streptomycin). HEK293T cells are maintained in DMEM with 10% dFBS, 10 mM HEPES, 0.1 mM NEAA and 100 U/mL Penicillin/100 µg/mL Streptomycin. SH-SY5Y cells are maintained in DMEM/F-12 medium supplemented with 10% dFBS and 100 U/mL Penicillin/100 µg/mL Streptomycin. HEK293 T-REx™ GFP-LRRK2 G2019S cells described in
Transduced cells in the 384-well assay plate were incubated with indicated inhibitors (5X, 5 µL/well) for 60 to 90 minutes. During this time, 6 X lysis buffer (Life Technologies) was supplemented with 30 nM Tb-labeled anti-LRRK2 pSer935 antibody and both protease and phosphatase inhibitor cocktails (Sigma). The complete 6 X lysis buffer was added to each well (5 µL/well) and the plates were incubated at room temperature in the dark for 2 hours. The plate was then read on an EnVision® multilabel plate reader (PerkinElmer, Waltham, MA) with excitation at 340 nm and emission 520 nm and 495 nm. Emission ratios of 520 nm/495 nm were calculated and plotted against the concentration of compound. A sigmoidal dose-response equation with varying slope was used to fit the data and generate IC50 values. Z′-factor values were calculated as: Z′-factor = 1 - [(3 × stdevno virus +3 × stdevmaxvirus)/(avgmaxvirus – avgno-virus)]. The IC50 values reported in
SH-SY5Y cells were transduced with 25% (v/v) BacMam LRRK2-GFP G2019S and plated (20 µL/well, 20,000 cells/well) onto eight 384-well assay plates as described above. 40 nL of 10 mM Tocris library compounds in DMSO were dispensed into each well of the assay plate by the ECHO system. Equal volumes of DMSO were dispensed into the control wells. Cells were incubated with compounds for 90 to 120 min prior to cell lysis and detection as described above. The percent inhibition is calculated as: [(average emission ratios of DMSO control wells – average emission ratios of compound wells)/(average emission ratios of DMSO control wells – average emission ratios of LRRK2-IN-1 treated wells)] * 100. For the dose-response follow-up studies, fresh compound DMSO stock solutions were prepared from powder. Curve fitting and IC50 value calculations were performed using XLFit4 software (IDBS) and a nonlinear regression equation for variable-slope sigmoidal dose response (model number 205). Z′-factor value was calculated as: Z′-factor = 1 - [(3 × stdevLRRK2-IN-1+3 × stdevDMSO)/(avgDMSO – avgLRRK2-IN-1)]. The IC50 values reported in
U-2 OS cells were transduced with BacMam LRRK2-GFP as described above. The cells were then plated on chambered coverslips (LabTek II/Nunc, Rochester, NY) at 50% confluency and incubated for 48 hours. The growth medium was replaced with phenol-red free DMEM® (Life Technologies, Carlsbad, CA) before live imaging. Cells were imaged under the same conditions for each mutant using an inverted microscope (Nikon Eclipse TE2000-S) equipped with a PlanApo 60X objective and a charge-coupled device camera (Diagnostic Instruments SPOT RT 2.3.0) using SPOT Advanced software.
Immunoblot analysis: BacMam LRRK2-GFP transduced U-2 OS or SH-SY5Y cells (107 cells) were left untreated or treated with 3 µM LRRK2-IN-I for 90 min. For lysate preparation, the cells were washed once with ice-cold PBS and lysed by addition of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 1% NP-40, 5 mM EDTA, 5 mM NaF, 150 mM NaCl, and 1∶100 of protease and phosphatase inhibitor cocktails). The lysate was cleared of debris by centrifugation. Equal amount of total protein was loaded onto each lane for SDS-PAGE (3–8% Tris Acetate). The proteins were transferred onto nitrocellulose membrane using the iBLOT® dry blotting system (Life Technologies) according to manufacturer’s protocol. The membranes were blocked and incubated with a primary antibody against LRRK2 (Cell Signaling Technologies), followed by incubation with alkaline phosphatase-conjugated secondary antibody (WesternBreeze® chromogenic Kit, Life Technologies). The blot was developed by using the chromogenic BCIP/NBT substrate (WesternBreeze® chromogenic Kit, Life Technologies). Immunoprecipitation analysis: HEK293 cells were induced to express GFP-LRRK2 G2019S by inclusion of doxycycline in the culture media at 1 µg/mL. Two days post induction, cells were treated for 90 minutes in the presence of 20 µM of compound or DMSO vehicle control. Cells were rapidly lysed in ice cold lysis buffer 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (w/v) 1 mM sodium orthovanadate, 10 mM sodium beta-glycerolphosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM benzamidine and 2 mM phenylmethanesulphonylfluoride (PMSF) and one tablet each complete protease inhibitor and PhosSTOP phosphatase inhibitor (Roche) and was supplemented with 1% (v/v) Triton X-100. GFP-LRRK2 was immunoprecipitated with GFP nanotrap beads (ChromoTek). Immunecomplexes were washed twice with lysis buffer supplemented with 300 mM NaCl and once with Buffer A (50 mM Tris/HCl, pH 7.5, 50 mM NaCl, 0.1 mM EGTA and 0.1% (v/v) 2-mercaptoethanol, and 0.27 M sucrose). Equal amounts of immunoprecipitated LRRK2 were fractionated on 4–12% Novex® NuPAGE® Bis-Tris gels and proteins were transferred to nitrocellulose. Blots were probed with phosphoserine 910, 935, 955 and 973 antibodies as described previously
The Biochemical LRRK2 TR-FRET activity assay was performed according to the manufacture recommended assay protocol (Life Technologies). Briefly, kinase reactions were performed in 10 µL volumes in black, low-volume 384-well plates (Corning #3676), with 400 nM Fluorescein-ERM (LRRKtide) substrate and 1% DMSO (residual from compound dilutions), in Kinase Buffer S (Life Technologies) supplemented with 2 mM DTT. Reactions performed with ATP equal to Km,app (134 µM) and with 580 ng/mL LRRK2 G2019S (Life Technologies). After incubation at room temperature for 1 hour, the kinase reactions were stopped by addition of 10 µL of 20 mM EDTA and 4 nM Tb-anti-pERM (pLRRKtide) antibody in TR-FRET dilution buffer (Life Technologies). After a 30 minute incubation at room temperature, TR-FRET measurements were obtained on a BMG PHERAstar plate reader using LanthaScreen® filters and settings. A 16 point, 2-fold dilution series of each compound was tested in triplicate at each concentration and assays were also performed in triplicate.
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We are grateful to Ryan Fischer for Tocris library compound dispensing. We would like to thank the Michael J. Fox for Parkinson’s Research for providing us with the LRRK2-IN-1 compound.