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: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: Detection of hLRRK2 and hLRRK2 R1441G expression and GTPase activity in Tg mice. A Real-time PCR assays of hLRRK2 and hLRRK2 R1441G mRNA expression in the brains of Tg mice. Data represent relative mRNA levels (mean ± SEM, n = 8) normalized to mouse apoB and are expressed in arbitrary units. B LRRK2 protein expression in the brains of non-Tg and Tg mice (n = 3). C hLRRK2 R1441G HEM and HOM mice lost less weight than non-Tg mice. Data are presented as the mean ± SEM (n = 6). D GTPase activity was measured using an enzyme-linked inorganic phosphate assay (ELIPA; n = 3). E Disruption of LRRK2 Ser935 phosphorylation in the brains of hLRRK2 , R1441G HOM mice. Total lysates from the brains of 12-month-old hLRRK2 R1441G HOM mice were analyzed by western blotting for phosphorylation of LRRK2 at Ser935; actin was used as a loading control. *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Disruption, Phospho-proteomics, Western Blot, Control

Journal: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: Locomotor activity in the open-field test was significantly reduced in hLRRK2 2 R1441G HOM mice. Data are presented as the mean ± SD (n = 5). *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques: Activity Assay

Journal: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: Several parameters of measured by the CatWalk system were affected in various ways in four mice. The swing velocity, cadence, and stride length were decreased in hLRRK2 R1441G HOM mice. In contrast, the stance duration and the pressure on the hind-paws BOS increased in hLRRK2 R1441G HOM mice. Data are presented as the mean ± SD (n = 5). *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques:

Journal: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: Comparison of [18F]FDOPA images from four groups of mice, showing significantly decreased uptake of the ligand among Tg mice: A coregistered coronal [ 18 F]FDOPA images of the non-Tg mouse striatum; B coregistered coronal [ 18 F]FDOPA images of the hLRRK2 mouse striatum; C coregistered coronal [ 18 F]FDOPA images of the hLRRK2 R1441G HEM mouse striatum; D coregistered coronal [ 18 F]FDOPA images of the hLRRK2 R1441G HOM mouse striatum; E average [ 18 F]FDOPA uptake in the region of interest (striatum) in various groups. The uptake values are A , B , C , and D for Group 1,2,3, and 4, respectively Data are presented as the mean ± SD (n = 5–6). *Significant effect, p < 0.05; **Significant effect, p < 0.01; ***Significant effect, p < 0.005; ****Significant effect, p < 0.001

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques: Comparison

Journal: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: Effect of SN in hLRRK2 , R1441G HEM and HOM mice SNc region. A Ultrastructural analysis of SN in hLRRK2 , R1441G HEM and HOM mice SNc region. Non-Tg and hLRRK2 represent a healthy mitochondrion; HEM and HOM represent a swollen mitochondrion. Selected regions in different magnification images (I, 10,000×; II, 20,000×). Asterisks indicate shrinkage and matrix condensation. HEM, R1441G HEM; HOM, R1441G HOM. B Western blot analysis of mitochondrial fission proteins in the mitochondrial fraction of the whole brain. Equal loading of the gel is demonstrated with Ponceau S staining for mitochondrial protein fragmentation performed on the blot before immunostaining. B Representative western blot for Drp1 and Fis1 expression. Densitometry of the Drp1 ( C ) and Fis1 ( D ) blots is shown as the fold increase (HEM or HOM hLRRK2 ). The figure shows a representative (of two) experiment. Values are means ± SD. *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques: Western Blot, Staining, Immunostaining, Expressing

Journal: Journal of Biomedical Science

Article Title: Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

doi: 10.1186/s12929-022-00844-9

Figure Lengend Snippet: LRRK2 R1441G HOM mice accumulated more autophagosomes in the SNc. A Transmission electron microscopic images of autophagosomes from the SN of 12-month-old hLRRK2 and R1441G transgenic mice. Selected regions in images at different magnifications (I, 10,000×; II, 20,000×). Arrows indicate autolysosomes, and asterisks indicate autophagosomes. Markers of autophagy (LC3 and p62) in the SNc of hLRRK2 and R1441G transgenic mice were determined by western blotting. p62 undergoes degradation at the early phase of autophagy. p62 in mitochondria serves as an adapter for autophagosome recognition. The data appear to downregulate the autophagy process, as observed by the increasing LC3-II conversion and the accumulation of p62, a marker of autophagic degradation

Article Snippet: Hemizygous (HEM) wild-type (WT) hLRRK2 mice (FVB/N-Tg ( LRRK2 )1Cjli/J, no. 009610) [ ], and HEM hLRRK2 R1441G transgenic (Tg) mice (FVB/N-Tg ( LRRK2 *R1441G)135Cjli/J, no. 009604) [ ] were purchased from the Jackson Laboratory and bred on an FVB/N background (Jackson stock number.

Techniques: Transmission Assay, Transgenic Assay, Western Blot, Marker

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 Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

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 Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

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 Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

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 Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

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 Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: In Vitro, Motility Assay, Concentration Assay