Abstract
Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson’s disease. In this study we compared the properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Unexpectedly, we strikingly find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open-closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.
Introduction
Autosomal dominant mutations that activate the LRRK2 (leucine rich repeat protein kinase-2) signalling pathway cause late onset Parkinson’s disease [1, 2]. LRRK2 is a large 2527 residue multidomain enzyme bearing an armadillo, ankyrin, leucine-rich repeats followed by a tandem Roco type GTPase consisting of a Roc and Cor domain, a Ser/Thr kinase domain and a C-terminal WD40 domain [3]. Various pathogenic mutations have been well-characterized that lie within the LRRK2 Roc (N1437H, R1441G/C/H), Cor (Y1699C), or kinase (G2019S, I2020T) domain [4]. The G2019S mutation is located within the conserved Mg2+ subdomain VII motif of the kinase domain and represents the most frequently observed mutation [5]. LRRK2 phosphorylates a subset of Rab GTPases, including Rab8A, Rab10 and Rab12 at a conserved Ser/Thr residue (Thr72 for Rab8A, Thr73 for Rab10, Ser106 for pRab12) [6, 7]. The LRRK2 phosphorylation site is located within the Rab effector-binding Switch-II motif. LRRK2 phosphorylated Rab proteins interact with new set of phosphobinding effectors such as RILPL1 and RILPL2 [6, 8].
All LRRK2 pathogenic mutations increase phosphorylation of Rab substrates including Rab10 [6, 7, 9]. These mutations also stimulate LRRK2 autophosphorylation at Ser1292 [10], but detection of this site is challenging especially for endogenous LRRK2, due to its low stoichiometry of phosphorylation. LRRK2 is also phosphorylated at several well studied serine residues that are termed biomarker sites including Ser910, Ser935, Ser955 and Ser973, located between the ankyrin and leucine rich repeats [11, 12]. Phosphorylation of Ser910 and Ser935 promotes interaction with 14-3-3 scaffolding proteins, preventing LRRK2 from assembling into inclusion like bodies in the cytosol [11, 13]. These biomarker sites have received much attention as they become dephosphorylated following pharmacological inhibition of LRRK2 [13]. Dephosphorylation of Ser935 in particular, has been widely exploited to assess in vivo efficacy of LRRK2 inhibitors [14–16]. It is unclear whether LRRK2 Ser910/Ser935 phosphorylation is regulated by autophosphorylation or via upstream kinases. Phosphorylation of the biomarker sites do not correlate with intrinsic LRRK2 kinase activity as mutation of these residues to Ala do not impact basal LRRK2 kinase activity [11, 17]. Furthermore, various pathogenic mutations including R1441C/G, Y1699C and I2020 mutations suppress the phosphorylation of Ser910 and Ser935 through unknown mechanisms [11, 13].
Evidence points towards LRRK2 controlling mitochondrial [18, 19] and lysosomal [20–24] homeostasis. Recent work has revealed that LRRK2 inhibits basal mitophagy in a variety of cells including dopaminergic neurons and that LRRK2 inhibitors promote mitophagy in LRRK2[G2019S] knock-in mice [25]. LRRK2 has also been shown to control repair of damaged endomembranes through its ability to phosphorylate Rab8A [26, 27]. Treatment of cells with agents that stress lysosomes, promote Rab protein phosphorylation by LRRK2 [28, 29]. Pathogenic LRRK2 also inhibits ciliogenesis in cholinergic interneurons through LRRK2 phosphorylated Rab10 forming a complex with RILPL1 [30, 31]. Cilia loss in cholinergic interneurons induced by LRRK2 pathogenic mutations may decrease ability of these cells to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons [30]. R1441G and I2020T pathogenic mutations as well as LRRK2 kinase inhibitors promote binding of LRRK2 to microtubule filaments [32–34]. Tomography and cryo-EM analysis suggest that pathogenic mutations induce the LRRK2 kinase domain to adopt a closed active conformation that becomes intrinsically capable of binding microtubules in a well-ordered and periodic manner [35, 36]. Furthermore, recruitment of LRRK2 to microtubules inhibits kinesin and dynein microtubule motility in biochemical studies [36].
There are two widely studied classes of kinase inhibitors namely, Type-I that bind to the kinase domain in a closed active conformation and Type-II that maintain the kinase in an open inactive-conformation [37]. It is likely that the widely utilized LRRK2 inhibitors are Type I compounds and would therefore induce LRRK2 to adopt a closed microtubule filament binding conformation [36]. The only Type II kinase inhibitor that have been reported to target LRRK2 are Ponatinib [38] and GZD-824 [36], compounds originally developed as inhibitors of the breakpoint cluster region-Abelson (Bcr-Abl) kinase to overcome clinically acquired mutation-induced resistance against imatinib the first line treatment of chronic myelogenous leukemia [39, 40]. Ponatinib and GZD-824 suppressed binding of LRRK2 to microtube filaments, consistent with these compounds interaction with LRRK2 in an open conformation [36].
In this study we compare the biochemical and in vivo properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824) LRRK2 kinase inhibitors. We find that Type II inhibitors similar to Type I compounds, suppress LRRK2 mediated phosphorylation of Rab proteins and stimulate basal mitophagy. Unexpectedly and in contrast to Type I inhibitors, Ponatinib and GZD-824 do not induce the dephosphorylation of LRRK2 at Ser935 and other nearby residues. We also find that Type II inhibitors suppress wild type LRRK2 more potently than pathogenic forms of this enzyme. These observations provide a framework to develop and characterize more selective Type II LRRK2 inhibitors as tools to dissect LRRK2 signaling functions.
Materials and methods
Materials
GSK3357679A [25] was synthesized at GlaxoSmithKline. MLi-2 was synthesized by Natalia Shpiro (University of Dundee) as described previously [16]. GZD-824 (#21508) was purchased from Cambridge Bioscience and Ponatinib (#4274) from Tocris. Microcystin-LR was purchased from Enzo Life Sciences (#ALX-350-012-M001). Human recombinant full length wild type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) were from ThermoFisher Scientific. Diisopropylfluorophosphate was from Sigma (#D0879).
Antibodies
Rabbit monoclonal antibodies for LRRK2 phospho-Ser935 (#UDD2) and phospho-Ser910 (#UDD1) were expressed and purified at University of Dundee as described previously [41]. The C-terminal total LRRK2 mouse monoclonal antibody was from Neuromab (clone N241A/34, #75-253). Rabbit monoclonal antibodies for LRRK2 phospho-Ser1292 (#ab203181), phospho-Ser955 (#ab169521), phospho-Ser973 (#ab181364), MJFF-phospho-Thr1357-LRRK2 (#ab270606) and the recombinant MJFF-phospho-Thr73-Rab10 (#ab230261) [42], MJFF-Rab10 total (#ab237703) and MJFF-phospho-Ser106-Rab12 (#ab256487) rabbit monoclonal antibodies were purchased from Abcam. The total Rab10 mouse antibody was from nanoTools (0680–100/Rab10-605B11, www.nanotools.de) [42]. The total Rab12 sheep polyclonal antibody was generated at University of Dundee, MRC PPU Reagents and Services (1st bleed, #SA224). Total Rab12 rabbit polyclonal antibody was from Proteintech (#18843-1-AP). Anti HA rat monoclonal antibody was purchased from Sigma-Roche (clone 3F10, #11867423001). The anti (pan)14-3-3 rabbit polyclonal antibody (#8312) and anti α-tubulin mouse monoclonal antibody (clone DM1A, mAb #3873) were purchased from Cell Signaling Technology. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Santa Cruz Biotechnology (#sc-32233).
Plasmids and transient transfections
cDNA constructs for overexpression of human LRRK2 and Rabs employed in the present study were the following: FLAG-LRRK2-WT (DU6841), HA-Rab10 (DU44250), HA-Rab29 (DU50222), HA-empty (DU44059), GFP-LRRK2 WT (DU13633), GFP-empty (DU13156) and were generated at the University of Dundee, MRC PPU Reagents and Services and are available on request (https://mrcppureagents.dundee.ac.uk/). Plasmids were amplified in Escherichia coli DH5a and purified using HiSpeed Plasmid Maxi Kit (Qiagen).
Cell cultures, treatments and lysis
Wild-type and homozygous LRRK2 [R1441C] [6] and LRRK2[G2019S] knock-in MEF [7] cells were isolated from littermate-matched mouse embryos at day E12.5 as described previously. MEFs were grown in Dulbecco’s modified Eagle medium (DMEM GIBCO, #11960-085) containing 10% (by vol) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin supplemented with non-essential amino acids and 1 mM sodium pyruvate. Human embryonic kidney 293 (HEK293) (#ATCC CRL-1573) and HeLa (#ATCC CCL-2) cells were purchased from American Tissue Culture Collection (ATCC) and cultured in DMEM containing 10% (by vol) FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cells were grown at 37°C, 5% (by vol) CO2 in a humidified atmosphere and regularly tested for mycoplasma contamination. The studies undertaken to produce these cells were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and regulations set by the University of Dundee and the U.K. Home Office. Animal studies and breeding were approved by the University of Dundee ethical committee and performed under a U.K. Home Office project license. The wild type and homozygous LRRK2[G201S] human lymphoblastoid cell lines were described previously [13] and maintained in suspension culture in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at cell density of 0.3 × 106-2 × 106 cells per ml. For experiments cultures of 3.5-5 million cells were utilized.
Inhibitor treatments
Inhibitors were dissolved in dimethyl sulfoxide (DMSO) (Sigma, #D2650). The inhibitors were diluted 1000x into cell medium. The equivalent volume of DMSO was added to the minus inhibitor controls. Experiments were performed in 6-well plates, 10 cm dishes,15 cm dishes or 15 ml Falcon-like tubes depending on the amount of cell extract required.
Transient transfections
Transient transfections were performed for 24 h using the linear polyethylenimine (PEI, Polyscience, #24765) method [43] with a final concentration of 2 μg/ml PEI and 6 μg of DNA for a 10 cm diameter dish with 10 ml of medium.
Cell Lysis
Cells were lysed in an ice-cold lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1% (by vol) Triton X-100, 10% (by vol) glycerol, 150 mM NaCl, 1 mM sodium orthovanadate, 50 mM NaF, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 1 μg/ml microcystin-LR, and cOmplete EDTA-free protease inhibitor cocktail (Roche, #11836170001). Lysates were incubated on ice for 30 minutes and clarified by centrifugation at 20800 x g at 4°C for 20 min. Supernatants were quantified by Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate #5000006) and samples prepared in 1x lithium dodecyl sulfate sample buffer final concentration (NuPage LDS Sample buffer 4x Invitrogen, #NP0008). When not deployed immediately, lysates were snap-frozen in liquid nitrogen and stored at −80 °C until use.
LRRK2 in vitro kinase assays
Kinase assays were performed in a 25 μl final reaction mixture containing immunoprecipitated HA-Rab10 conjugated to HA-agarose beads (Monoclonal Anti HA-agarose, clone HA-7, Sigma, #A2095), recombinant full length LRRK2[G2019S] (10 nM, 50 ng) and 50 mM Tris–HCl pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 1 mM ATP in the presence or absence of the indicated concentration of inhibitor. The Rab10 was expressed by transient transfection in HEK293 cells. HEK293 cells were treated with 500 μM MLi-2 LRRK2 inhibitor for 90 min prior to cell lysis to ensure Rab10 is dephosphorylated. For each assay the Rab10 protein was immunoprecipitated from 0.5 mg of HEK293 cell lysate. After 2.5 h of incubation at +4°C in rotation, immunoprecipitates were washed with lysis buffer supplemented with 0.2 M NaCl, then twice with Lysis buffer and finally washed with 50 mM Tris HCl pH 7.5. Immunoprecipitates were immediately used for the kinase assay without snap freezing. The kinase reactions were carried out at 30 °C with shaking at 1500 rpm, and at the indicated time points the reactions were terminated by the addition of 25 μl of 2x LDS containing 1% (by vol) 2-mercaptoethanol and heated for 70 °C for 10 min. The reactions were centrifuged through a 0.22 μm Spin X filter (Costar, # CLS8161) and supernatant stored at −80 °C prior to immunoblotting. One third of final reaction volume was used for immunoblotting analysis.
IC50 LRRK2 Nictide assay
Reactions were undertaken with wild type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific. Peptide kinase assays, performed in triplicate, were set up in a total volume of 25.5 μL containing 13.7 ng LRRK2 kinase in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 20 μM Nictide (an optimized peptide substrate for LRRK2 [44]), 0.1 μM [γ-33P]ATP (~500 cpm/pmol) and the indicated concentrations of inhibitor dissolved in DMSO. After incubation for 30 min at room temperature °C, reactions were terminated by harvesting the whole reaction volume onto P81 phosphocellulose filter plate and immersion in 50 mM phosphoric acid. Samples were washed extensively and the incorporation of [γ-33P]ATP into Nictide was quantified by Cerenkov counting. IC50 values were calculated with GraphPad Prism using non-linear regression analysis.
Kinase Profiling
Protein kinase profiling of the Type I (GSK3357679A and MLi-2) and Type II inhibitors (GZD-824 and Ponatinib) were undertaken at a concentration of 0.1 and 1 μM and carried out against the Dundee panel of 140 protein kinases at the International Centre for Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk). The kinase profiling of the each inhibitor was undertaken at a concentration of 1 and/or 10 μM. Results for each kinase are presented as the mean kinase activity ± S.D. for an assay undertaken in duplicate relative to a control kinase assay in which the inhibitor was omitted. Abbreviations and assay conditions used for each kinase are available (http://www.kinase-screen.mrc.ac.uk/services/premier-screen).
Quantitative immunoblot analysis
Cell extracts were mixed with a quarter of a volume of NuPage LDS Sample buffer 4x Invitrogen and heated at 95 °C for 5 minutes. 10-30 μg of samples were loaded onto NuPAGE 4–12% Bis–Tris Midi Gel (Thermo Fisher Scientific, Cat# WG1403BOX) and electrophoresed with the NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, Cat# NP0001-02) at 90 V. After 30 min the voltage was increased at 120 V for 1 h 30 min. At the end of electrophoresis, proteins were electrophoretically transferred onto the nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 μm NC) at 100 V for 90 min on ice in the transfer buffer (48 mM Tris–HCl and 39 mM glycine supplemented with 20% methanol). Transferred membrane was blocked with 5% (w/v) skim milk powder dissolved in TBS-T [20 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.1% (v/v) Tween 20] at room temperature for 1 h. The membrane was typically cropped into three pieces, namely the ‘top piece’ (from the top of the membrane to 75 kDa), the ‘middle piece’ (between 75 and 30 kDa) and the ‘bottom piece’ (from 30 kDa to the bottom of the membrane). All primary antibodies were used at 1 μg/ml final concentration and incubated in TBS-T containing 5% (by mass) bovine serum albumin with exception of α-tubulin and GAPDH antibodies that were diluted 1:5000 and 1:2000 respectively. The top piece was incubated with the indicated rabbit anti-LRRK2 phosphorylation site antibody multiplexed with mouse anti-LRRK2 C-terminus total antibody. The middle piece was incubated with mouse anti-GAPDH or anti-α-tubulin antibody. The bottom pieces were incubated with indicated total and phospho-Rab antibodies. Membranes were incubated in primary antibody overnight at 4°C. Prior to secondary antibody incubation, membranes were washed three times with TBS-T for 10 min each. The top and bottom pieces were incubated with goat anti-mouse IRDye 680LT (#926-68020) secondary antibody multiplexed with goat anti-rabbit IRDye 800CW (#926-32211) secondary antibody diluted in TBS-T (1:25000 dilution) for 1 h at room temperature. The middle piece was incubated with goat anti-mouse IRDye 800CW (#926-32210) secondary antibody diluted in TBS-T (1:25000 dilution) at room temperature for 1 h. Membranes were washed with TBS-T for three times with a 10 min incubation for each wash. Protein bands were acquired via near infrared fluorescent detection using the Odyssey CLx imaging system and quantified using the Image Studio software.
Analysis of 14-3-3 binding
This was undertaken as described previously [11]. Briefly, wild type FLAG-LRRK2 was transiently expressed in HEK293 cells with HA-Rab29. 24h post-transfection cells were lysed with lysis buffer and snap frozen. LRRK2 was immunoprecipitated from 4 mg of lysate using 20 μl anti-FLAG-M2 agarose beads (Sigma, #A2220) for 2 h in rotation at +4°C. Immunoprecipitates were washed once with lysis buffer supplemented with 0.5% (by vol) Nonidet P-40 (instead of Triton X-100) and 0.5 M NaCl followed by two washes. Samples were eluted in 2x LDS sample buffer (20 μl per 10 μl of resin) and heated at 70 °C for 10 min. After centrifugation through 0.22 μm Spin X filter, the eluted protein was subjected to immunoblotting with the indicted antibodies. 10 μl of the final reaction mixture was needed for the detection of binding of endogenous 14-3-3 and 1 μl for detection of LRRK2.
Immunofluorescence microscopy
HeLa cells were seeded on coverslips and transfected with the plasmid reported in each image 24 h before fixation. Following the treatments with the inhibitors described in figure legends, cells were washed twice in DPBS and fixed with 3.7% Paraformaldehyde pH=7.3 (Agar scientific, #R1026), 200 mM HEPES, for 20 minutes. Cells were then washed twice with DMEM, 20mM HEPES, and then incubated for 10 minutes with DMEM, 10mM HEPES. After two washes in DPBS, fixed cells were permeabilized with 0.2% (by vol) NP-40 in DPBS for 10 min. Cells were blocked using 1% (by mass) BSA in DPBS, then incubated for 1 h with primary antibodies diluted in 1% (by mass) BSA in DPBS, washed three times in 0.2% (by mass) BSA in DPBS, and incubated for 1 h with secondary antibodies. The coverslips were washed three times with 0.2% (by mass) BSA in PBS. Coverslips were washed once more in double distilled water and mounted on slides (VWR, #631-0909) using ProLong™ Diamond Antifade Mountant (ThermoFisher, #P36961). For localization of the Golgi compartment, Golgin-97 was stained with rabbit anti-GM97 (CST, #D8P2K) and visualized with goat antirabbit conjugated to Alexa Fluor 405 (Life Technologies, #P10994). HA-Rab29 was stained with mouse anti-HA (Abcam, clone HA.C5 #ab18181) and visualized with goat anti-mouse conjugated to Alexa Fluor 594 (Life Technologies, #A-11032). The images were collected on an LSM710 laser scanning confocal microscope (Carl Zeiss) using the Å~63 Plan-Apochromat objective (NA 1.4).
Human neutrophils isolation
Neutrophils were isolated from 20 ml of whole blood collected from two healthy donors using an immune-magnetic negative isolation described previously [45]. 5 million cells from each condition were re-suspended in 1 ml of RPMI medium at and treated the indicated inhibitors or DMSO as a control for 45 min at room temperature. Cells were pelleted by centrifugation at 335xg for 5 min, the supernatants decanted, and the pellets lysed in lysis buffer freshly supplemented with 0.5 mM diisopropylfluorophosphate to inhibit high levels of intrinsic serine protease activity present in neutrophils [45]. Neutrophil lysates were incubated on ice for 30 minutes, centrifuged at 20800 x g for 20 min at 4 °C, snap-frozen in liquid nitrogen and stored at −80 °C. The study was approved by the independent ethics committee of the University of Dundee in the United Kingdom. All participants gave written informed consent.
Mito-QC primary mouse embryonic fibroblasts culture
To assess mitophagy, wild type, G2019S knock in, and LRRK2 knock out primary mouse embryonic fibroblasts (MEFs) were derived, from time-mated pregnant females at E12.5 as previously described [25]. Briefly, cells were plated on glass coverslips and incubated with DMEM (Gibco, 11960-044) supplemented with 10% FBS, 2 mM L-Glutamine (Gibco, 2503-081), 1% Non-essential amino acids (Gibco, 11140-035), 1% Antibiotics (Penicillin/Streptomycin 100 U/ml penicillin and 100 μg/ml streptomycin; Gibco), and 150μM β-Mercaptoethanol (Gibco, 21985-023) at 37ºC under a humidified 5% CO2 atmosphere. MEFs were treated for 24 hours with GZD-824 (3, 10, 30, 100, 300 nM). GSK3357679A 100nM (Ding et al., in prep) was used as a positive control. At the end of the treatment, MEFs were briefly washed twice in DPBS (Gibco, 14190-094), and fixed in 3.7% Paraformaldehyde pH=7.00 (Sigma, P6148), 200 mM HEPES, for 20 minutes. Cells were then washed two times with DMEM, 10mM HEPES, and then incubated for 10 minutes with DMEM, 10mM HEPES. Cells were washed in DPBS and mounted on a slide (VWR, Superfrost, #631-0909) with Prolong Diamond (Thermo Fisher Scientific, #P36961). Images were acquired using a Nikon Eclipse Ti-S fluorescence microscope with a 63x objective. Images were analysed using the mito-QC counter as previously described (https://doi.org/10.1016/j.mad.2019.111196). Experiments were performed in 3-4 independent experiments and data was analysed with a one-way ANOVA followed by a Dunnett’s multiple comparison test using the GraphPad Prism software (version 8.4.3 (686)).
Results
Type-II inhibitors target wild type LRRK2 with greater potency than pathogenic LRRK2[G2019S] in vitro
We first assessed the in vitro potency of GZD-824 and Ponatinib towards full length wild type and G2019S pathogenic LRRK2 mutant in a biochemical assay using the synthetic peptide substrate termed Nictide [44] at an ATP concentration of 100 μM. This revealed that GZD-824 inhibited wild type LRRK2 and LRRK2[G2019S] with an IC50 of 17 nM and 80 nM respectively. Ponatinib was ~ 5-fold weaker inhibitor than GZD-824. Ponatinib also targeted wild type LRRK2 (IC50 of 100 nM) ~4-fold more potently than LRRK2[G2019S] (IC50 of 400 nM) (Table 1). The two type 1 LRRK2 kinase inhibitors used in this study, namely GSK3357679A and MLi-2, inhibited LRRK2 with sub-nanomolar potency with MLi-2 inhibiting LRRK2[G2019S] ~2-fold more strongly than wild type LRRK2. GSK3357679A displayed similar potency towards wild type LRRK2 and LRRK2[G2019S (Table 1). We also assessed the selectivity of GZD-824 (Fig S1A) and Ponatinib (Fig S1B) towards a panel of 140 protein kinases. This revealed that GZD-824 is not selective for LRRK2 and inhibited numerous other serine/threonine as well as tyrosine kinases. At 0.1 and 1.0 μM, GZD-824 suppressed the activity of 22 and 43 of the 140 kinases assayed by over 80% respectively (Fig S1A). The kinase selectivity of Ponatinib has also been assessed previously and found to inhibit 38 kinases out of 100 kinases tested with and IC50 of <10 nM, indicating that like GZD-824 it is a multi-kinase inhibitor [39]. By contrast, GSK3357679A (Fig S1C) and MLi-2 [16] are highly selective kinase inhibitors.
Type-II LRRK2 inhibitors suppress LRRK2 mediated Rab10 phosphorylation without suppressing Ser935 phosphorylation
We assessed the ability of GZD-824 and Ponatinib to inhibit LRRK2 mediated phosphorylation of Rab10 at Thr73 in LRRK2 wild type mouse embryonic fibroblast cells (MEFs). Cells were pre-incubated for 2 h with the indicated doses of GZD-824 or DMSO as a control and phosphorylation of Rab10 (Thr73), Rab12 (Ser106) and LRRK2 at Ser935 monitored by quantitative phospho-immunoblotting. GZD-824 induced a dose dependent inhibition of Rab10 and Rab12 phosphorylation with an IC50 of ~300 nM (Fig 1A). 1 μM GZD-824 reduced Rab10 and Rab12 phosphorylation to almost background levels similar to that observed with the MLi-2 Type 1 Inhibitor [16]. Strikingly, GZD-824 did not induce dephosphorylation of LRRK2 at Ser935, under conditions which MLi-2 induced complete dephosphorylation of this site (Fig 1A). Increasing GZD-824 concentrations to 10 μM did not reduce LRRK2 phosphorylation at Ser935 significantly (SFig2A). We observed that concentrations of 10 μM or higher GZD-824 impacted cell morphology and attachment, so we employed GZD-824 at 5 μM or less in subsequent studies. Ponatinib was a weaker inhibitor, and only partially reduced Rab10 phosphorylation ~50% inhibition at 10 μM, also without affecting Ser935 phosphorylation (Fig 1B). Concentrations of Ponatinib above 10 μM were also observed to impact cell morphology and attachment. GZD-824 suppressed Rab10 and Rab12 phosphorylation in a time dependent manner, with a 50% reduction observed within ~10 min (Fig 1C). Rab10 and Rab12 phosphorylation remained suppressed and Ser935 phosphorylation unaffected over a 24 h time course of 1 μM GZD-824 treatment (Fig 1D). GZD-824 also inhibited Rab10 phosphorylation in a dose dependent manner in primary human neutrophils isolated from two donors with maximal inhibition observed at 300 nM GZD-824 (Fig 2). Phosphorylation of Ser935 as well as phosphorylation of other biomarker sites Ser910, Ser955 and Ser973 was unaffected by concentration of GZD-824 up to 3 μM (Fig 2).
GZD-824 inhibits pathogenic LRRK2[R1441C] and LRRK2[G2019S] with lower potency than wild type LRRK2
We next compared the potency at which GZD-824 suppressed Rab10 phosphorylation in wild type and LRRK2[R1441C] (Fig 3A) as well as LRRK2[G2019S] knock-in MEFs (Fig 3B). In these experiments we observed that higher doses of GZD-824 were required to reduce phosphorylation of Rab10 in the pathogenic mutant cells. We estimate that the IC50 for dephosphorylation of Rab10 in wild type, R1441C and G2019S MEFs is 70 nM, 262 nM and 233 nM respectively. 1 μM GZD-824 reduced Rab10 phosphorylation to almost basal levels in wild type cells, but only reduced Rab10 phosphorylation ~ 3 to 3.5-fold in LRRK2[R1441C] MEFs and LRRK2[G2019S] MEFs. Earlier work revealed that the LRRK2[R1441C] mutation reduces LRRK2 Ser935 phosphorylation [11]. Consistent with this, we observed reduced phosphorylation of LRRK2 at Ser935 in the LRRK2[R1441C] knock-in cell lines (Fig 3A, 3C, 3D). Treatment with GZD-824 rescued Ser935 phosphorylation in LRRK2[R1441C] knock-in cells in a dose (Fig 3A) and time dependent manner (Fig 3C, 3D) to levels similar to that observed in wild type cells (Fig 3A, 3C, 3D). Time course analysis revealed that 1 μM GZD-824 increased Ser935 phosphorylation in LRRK2[R1441C] cells by 40 min plateauing at 160 min (Fig 3C, 3D). 1 μM GZD-824 only partially reduced by ~2-fold Rab10 phosphorylation in LRRK2[R1441C] knock-in cells (Fig 3C, 3D). As reported previously [11], the G2019S mutation did not reduce Ser935 phosphorylation and treatment of these cells with GZD-824 did not impact Ser935 phosphorylation (Fig 3B).
GZD-824 does not inhibit 14-3-3-binding or Rab29 mediated LRRK2 recruitment to the Golgi
Phosphorylation of Ser910 and Ser935 regulates the ability of LRRK2 to interact with 14-3-3 adaptor proteins [11]. To study the impact that GZD-824 has on 14-3-3 binding, we treated HEK293 cells overexpressing wild type LRRK2 with 5 μM GZD-824 for 4 h, immunoprecipitated LRRK2 and immunoblotted for co-immunoprecipitated endogenous 14-3-3 using an antibody that picks up multiple human isoforms. We observed that GZD-824 had no significant impact on 14-3-3 binding under these conditions, whereas 1 μM GSK3357679A ablated 14-3-3 binding and Ser935 phosphorylation (Fig 4A).
Rab29 is localized at the Golgi and co-expression of LRRK2 and Rab29 in HeLa cells strikingly results in the bulk of LRRK2 being recruited to the Golgi apparatus (Fig 4B, SFig3–4) [46]. Treatment of HeLa cells co-expressing LRRK2 and Rab29 with 5 μM GZD-824 for 4 h did not significantly impact Rab29 mediated relocalization of LRRK2 to the Golgi apparatus (Fig 4B). In parallel experiments, treatment with 1 μM GSK3357679A for 4h also had no impact on recruitment of LRRK2 to the Golgi by Rab29 (Fig 4B). Control immunoblotting study for this experiment is shown in Fig 4C.
GZD-824 stimulates basal mitophagy similarly to GSK3357679A
Previous work revealed that pathogenic LRRK2[G2019S] mutation significantly reduces basal mitophagy, but not general autophagy, in primary MEFs and various mouse tissues in a manner that is rescued by inhibiting LRRK2 with structurally distinct and selective Type I inhibitors, GSK3357679A and MLi-2 [25]. We therefore monitored the impact that 10-300 nM GZD-824 had on basal mitophagy in previously generated wild type, LRRK2[G2019S] knock-in and LRRK2 knock-out MEFs [7] stably expressing the mito-QC mitophagy reporter [25]. The mito-QC reporter is localized to mitochondria via an outer mitochondrial membrane targeting sequence derived from the protein FIS1 and bears a tandem GFP and mCherry tag. When a mitochondrion is delivered to lysosomes, the low lysosomal luminal pH quenches the GFP but not the mCherry signal. The degree of mitophagy is determined by the appearance of mCherry-only puncta, which represent mitochondria engulfed into autolysosomes [47–49]. Treatment of wild type or LRRK2[G2019S] cells for 24 h with 30-300 nM GZD-824 stimulated basal mitophagy ~2-fold, to the same extent as GSK3357679A (Fig 5A, 5B). This effect was specific for inhibition of LRRK2, as neither GZD-824 or GSK3357679A impacted basal mitophagy in LRRK2 knock-out cells. Consistent with previous work [25], basal mitophagy was lower in LRRK2[G2019S] MEFs compared to wild type MEFs and treatment with Type I or Type II inhibitors increased basal mitophagy to levels similar to those observed in LRRK2 knock-out cells (Fig 5A, 5B).
Discussion
In this study we compared the in vitro and cellular properties of Type I and Type II LRRK2 inhibitors. Type II inhibitors suppressed LRRK2 protein kinase activity in cells as judged by their ability to inhibit LRRK2 mediated Rab protein phosphorylation and stimulate basal mitophagy similar to Type I compounds. The most striking difference between Type I and Type II compounds is that Type II inhibitors do not induce dephosphorylation of Ser935 or other LRRK2 biomarker phosphorylation sites. This suggests that Type II inhibitors preserve biomarker site phosphorylation on LRRK2 by maintaining the LRRK2 kinase domain in the open inactive conformation. The mechanism underlying this phenomenon is not known, but we hypothesize that in the Type II-open kinase domain conformation, the N-terminal biomarker sites on LRRK2 are exposed for phosphorylation by the upstream kinase(s) and/or rendered inaccessible to protein phosphatase(s). Vice versa, in the Type I-closed conformation, access of the N-terminal biomarker sites to upstream kinase(s) could be restricted/or N-terminal biomarker sites could be better exposed to protein phosphatase(s). In future analysis it would be important to compare how Type I and Type II inhibitors influence the conformation and accessibility of the biomarker sites to upstream kinases and phosphatases.
The finding that prolonged treatment of cells with GZD-824 does not impact on Ser935 phosphorylation under conditions where Rab10 phosphorylation remains suppressed, argues against the biomarker sites located in the N-terminal region of LRRK2 being controlled by autophosphorylation. Although distinct kinases have been proposed to phosphorylate these sites [50–52], further work is required to validate which kinase(s) target these residues in vivo. A protein phosphatase-1 (PP1) complex has been reported to dephosphorylate these sites in vivo [53]. Future work should also explore whether interaction of LRRK2 with microtubule is involved in controlling biomarker phosphorylation. Targeting of LRRK2 to microtubules in the closed kinase conformation, could bring LRRK2 in association with a microtubule associated phosphatase and/or restrict access to an upstream protein kinase.
Our conclusions are also consistent with the finding that the majority of pathogenic mutations that promote the active closed conformation of LRRK2 (R1441G/C, Y1699C, I2020T) suppress biomarker site phosphorylation [11]. Consistent with this, treatment of LRRK2[R1441C] knock-in MEFs with GZD-824, would be predicted to induce the open conformation and thus rescue Ser935 phosphorylation and this is what we observe (Fig 3). Our results emphasize that the steady state phosphorylation of the biomarker sites on LRRK2 correlates with the open-closed conformation of the LRRK2 kinase domain rather than the intrinsic kinase activity towards Rab substrates. The finding that treatment of cells with Type II inhibitors does not further increase biomarker site phosphorylation suggests that the bulk of the cellular LRRK2 is in the open inactive conformation.
Neither GZD-824 (Fig S1A) or Ponatinib [39] are selective kinase inhibitors (Fig S1). Ponatinib is clinically approved for treating chronic myeloid leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia [54]. GZD-824 and Ponatinib inhibits wild type LRRK2 more potently than pathogenic LRRK2 in vitro (Table 1) and in vivo (Fig 1), a feature that will likely be similar for other Type II inhibitors. As pathogenic mutations favor the closed conformation of LRRK2 kinase domain, it would be expected that a higher concentration of a Type II inhibitor would be required to inhibit pathogenic LRRK2. This difference in potency between wild type and pathogenic LRRK2 for Type II inhibitors would need to be taken into consideration when evaluating the optimal therapeutic doses of a potential future Type II inhibitor compound for treating LRRK2 driven Parkinson’s disease.
To our knowledge all of the well-studied LRRK2 inhibitors induce dephosphorylation of the N-terminal biomarker sites on LRRK2 suggesting that these inhibitors will be Type I compounds. Previous work in LRRK2-deficient rodents as well as nonhuman primates treated with LRRK2 inhibitors highlighted preclinical liability concerns in lung and kidney, resulting from loss of LRRK2 kinase activity that might be associated with altered autophagy and lysosome biology [55–57]. In future work it would be important to explore the physiological impact that selective Type II LRRK2 inhibitors have on animal models in comparison to Type I compounds. It is possible that Type II compounds that do not induce microtubule association or lead to dephosphorylation of biomarker sites may have a different physiological impact. The poor selectivity of GZD-824 and Ponatinib precludes their use as a tool compounds to better probe the physiological impacts of Type II LRRK2 inhibitors. In future work it would therefore be important to develop more selective Type II LRRK2 tool inhibitors to enable their properties to be better studied.
Funding
This work was supported by the Medical Research Council [grant numbers MC_UU_12016/2 (D.R.A.); MC_UU_00018/2 (I.G.G)] and GlaxoSmithKline as part of the Division of Signal Transduction Therapy Consortium.
Data availability
All of the primary data that is presented in this study can be requested in electronic form by contacting Dario Alessi (d.r.alessi{at}dundee.ac.uk). All plasmids and antibodies (and associated datasheets) generated at the MRC Protein Phosphorylation and Ubiquitylation Unit at the University of Dundee can be requested through our reagent’s website https://mrcppureagents.dundee.ac.uk/.
Author Contributions
A.T designed, executed all experiments in this study presented in Figures 1, 2, 3, 4, Supplementary Figure 2A, 3, 4 analyzed and interpreted data and preparation of the manuscript. F.S. designed and executed and interpreted experiments relating to mitophagy analysis in Fig 5. I.G.G. oversaw studies relating to autophagy analyses. A.D.R was involved with experimental design, analysis, interpretation of data and preparation of the manuscript. D.R.A. helped with experimental design, analysis and interpretation of data and preparation of the manuscript.
Acknowledgements
We thank Paul Davies for helpful discussion, Esther Sammler for preparing human neutrophils and the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Gary Hunter), the MRC-PPU tissue culture team (coordinated by Edwin Allen), MRC PPU Reagents & Services antibody and protein purification teams (coordinated by Hilary McLauchlan and James Hastie) and kinase profiling (coordinated by Jennifer Moran).
Footnotes
This version of the manuscript has an updated version of Fig 3A, 3B and Fig 4A