Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: In Vitro, Motility Assay, Concentration Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: Structures of the LRRK2 Type II inhibitors employed in this study.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques:

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: Type II inhibitors inhibit wild-type LRRK2 with greater potency than pathogenic LRRK2[G2019S] in vitro

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: In Vitro

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A , B ) Wild-type MEFs were treated with or without the indicated concentrations of inhibitors for 2 h. Cells were lysed, and 20 µg of extract was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different dish of cells (three replicates per condition). The membranes were developed using the Odyssey CLx Western Blot imaging system. ( C ) As in ( A ) except that cells were treated with 1 μM GZD-824 or 100 nM MLi-2 for the indicated times. ( D ) As in ( A ) except that cells were treated with 1 μM GZD-824 or 100 nM MLi-2 or 100 nM GSK3357679A for 24 h. ( A – D ) Immunoblots were quantified using the Image Studio software. Data are presented relative to the phosphorylation ratio observed in cells treated with DMSO (no inhibitor), as mean ± SD. ( A – C ) SD are derived from the replicates show in in the presented blots. ( D ) SD are derived from duplicates run in independent gels.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Western Blot, Imaging, Software, Derivative Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A , B ) The indicated littermate matched wild type and LRRK2 pathogenic knock-in MEFs (passages n. 15–16) were treated with or without the indicated concentrations of inhibitors for 2 h. Cells were lysed, and 25 µg of extract was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different dish of cells. The membranes were developed using the Odyssey CLx Western Blot imaging system. ( C , D ) As in ( A ) except that cells were treated with 1 μM GZD-824 or 100 nM MLi-2 for the indicated times. Each lane represents cell extract obtained from a different dish of cells (two replicates per condition). ( A – D ) Immunoblots were quantified using the Image Studio software. Data are presented relative to the phosphorylation ratio observed in cells treated with DMSO (no inhibitor), as mean ± SD. ( A , B ) SD are derived from duplicates run in independent gels. ( C , D ) SD are derived from the replicates show in the presented blots. IC 50 values were calculated with GraphPad Prism (version 9.1.0) using non-linear regression analysis.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Knock-In, Western Blot, Imaging, Software, Derivative Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A , B ) The indicated littermate matched wild type and LRRK2 pathogenic knock-in MEFs (passages n. 15–16) were treated with or without the indicated concentrations of inhibitors for 2 h. Cells were lysed, and 25 µg of extract was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different dish of cells. The membranes were developed using the Odyssey CLx Western Blot imaging system. Immunoblots were quantified using the Image Studio software. Data are presented relative to the phosphorylation ratio observed in cells treated with DMSO (no inhibitor), as mean ± SD. ( A , B ) SD are derived from duplicates run in independent gels. IC 50 values were calculated with GraphPad Prism (version 9.1.0) using non-linear regression analysis.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Knock-In, Western Blot, Imaging, Software, Derivative Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A , C ) Inhibitor resistant immortalized MEFs LRRK2 [A2016] were treated with or without the indicated concentrations of inhibitors for 2 h. Cells were lysed, and 20 µg of extract was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different dish of cells (three replicates per condition). ( B , D ) As in ( A , C ) except that the cells were wild type. The membranes were developed using the Odyssey CLx Western Blot imaging system. Immunoblots were quantified using the Image Studio software. Data are presented relative to the phosphorylation ratio observed in cells treated with DMSO (no inhibitor), as mean ± SD. ( A , B ) SD are derived from the replicates show in the presented blots.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Western Blot, Imaging, Software, Derivative Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A ) HEK293 cells were transiently transfected for 24 h with the indicated constructs. 24 h post-transfection cells were treated with the indicated concentrations of inhibitors for the indicated times. An amount of 15 µg of whole cell extracts was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Flag-LRRK2 is immunoprecipitated and subjected to immunoblotting with the indicated antibodies. Each lane represents cell extract obtained from a different dish of cells. The membranes were developed using the Odyssey CLx Western Blot imaging system. ( B ) HeLa cells were transiently transfected with wild-type GFP-LRRK2 in the presence of absence of HA-Rab29. Twenty-four hours post-transfection cells were treated ± the indicated concentration of inhibitor for 4 h. Cells were fixed in 3.7% (by vol) paraformaldehyde and stained with mouse anti-HA and anti-Golgin-97 (trans Golgi marker). Scale bar represents 10 µm. The Figures are representative of at least two independent experiments. ( C ) HeLa cells were transiently transfected for 24 h with the indicated constructs. 24 h post-transfection cells were treated with the indicated concentrations of inhibitors for the indicated times. An amount of 15 µg of whole cell extracts was subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different dish of cells. The membranes were developed using the Odyssey CLx Western Blot imaging system ( A , C ) Immunoblots were quantified using the Image Studio software. Data are presented relative to the phosphorylation ratio observed in cells treated with DMSO (no inhibitor), as mean ± SD. ( A , C ) SD are derived from the replicates shown in the presented blots.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Transfection, Construct, Western Blot, Immunoprecipitation, Imaging, Concentration Assay, Staining, Marker, Software, Derivative Assay

Journal: Biochemical Journal

Article Title: Impact of Type II LRRK2 inhibitors on signaling and mitophagy

doi: 10.1042/BCJ20210375

Figure Lengend Snippet: ( A ) Representative images and corresponding mitophagy mask generated with the mito-QC counter of wild-type, homozygous LRRK2 G2019S, and LRRK2 KO primary mito-QC MEFs. Cells were treated with or without the indicated concentrations of inhibitors for 24 h. ( B ) Quantitation of mitophagy in cells treated with incremental doses of GZD-824. Data are represented as the mean ± SEM of 3–4 experiments and was analyzed with a one-way ANOVA followed by a Dunnett's multiple comparison test. * P < 0.05, ** P < 0.01.

Article Snippet: Reactions were undertaken with wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) purchased from ThermoFisher Scientific.

Techniques: Generated, Quantitation Assay