Impact of Type II LRRK2 inhibitors on signalling and mitophagy
Authors/Creators
- 1. Medical Research Council (MRC) Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee
- 2. GlaxoSmithKline Pharmaceuticals R&D, Medicines Research Centre
Description
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 first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824, Rebastinib and Ponatinib) 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. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we 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. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open- vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggestings this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.
FILE DESCRIPTIONS
The blots shown in the Figures were developed using the two fluorescent channels available in LI-COR (800 nM, green – 700 nM, red). Different contrasts of the same membranes have been uploaded.
Figure 2
Figure_2A-quantitations.xls: raw data for charts in Figure 2A
Figure_2A_700_1-LRRK2_Rab10_totals_GAPDH.png
Top to bottom
1: total LRRK2 blot shown in Figure 2A
2: GPDH loading control for blots in Figure 2A and total Rab10 shown in Figure 2A. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa
Figure_2A_700_2-Rab12_total.png
Total Rab12 shown in Figure 2A
Figure_2A_700_3-tubulin.png
Top to bottom
3: alpha-tubulin loading control for blots in Figure 2A
Figure_2A_800_1-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 2A
2: Rab10 pThr73 blot shown in Figure 2A
Figure_2A_800_2-Rab12pS105.png
Phospho-Rab12 pSer105 shown in Figure 2A
Figure_2B-quantitations.xls: raw data for charts in Figure 2B
Figure_2B_700_1-LRRK2_Rab10_totals_GAPDH_tubulin.png
Top to bottom
1: total LRRK2 blot shown in Figure 2B
2: GPDH loading control for blots in Figure 2B and total Rab10 shown in Figure 2B. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa
3: alpha-tubulin loading control for blots in Figure 2B
Figure_2B_700_2-Rab12_total.png
Total Rab12 shown in Figure 2B
Figure-2B_800_1-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 2B
2: Rab10 pThr73 blot shown in Figure 2B
Figure-2B_800_2-Rab12pS105.png
Phospho-Rab12 pSer105 shown in Figure 2B
Figure_2C-quantitations: raw data for charts in Figure 2C
Figure_2C_700_1-LRRK2_Rab10_totals_GAPDH.png
Top to bottom
1: total LRRK2 blot shown in Figure 2C
2: GPDH loading control for blots in Figure 2C and total Rab10 shown in Figure 2C. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa
3: alpha-tubulin loading control for blots in Figure 2C – higher contrast of Figure-2C 700-3-alpha_tubulin – low contrast)
Figure_2C_700_2-Rab12_total.png
Total Rab12 shown in Figure 2C
Figure_2C_700_3-tubulin.png
Top to bottom
1-2: lower contrast of Figure-2C 680-1 LRRK2_Rab10_totals_GAPDH
3: alpha-tubulin loading control for blots in Figure 2C
Figure_2C_800_1-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 2C
2: Rab10 pThr73 blot shown in Figure 2C
Figure_2C_800_2-Rab12pS105.png
Phospho-Rab12 pSer105 shown in Figure 2C
Figure_2D-quantitations.xls: raw data for charts in Figure 2D
Figure_2D_700_1-LRRK2_total_tubulin.png
Top to bottom
1: total LRRK2 blot shown in Figure 2D
2: lower contrast of total Rab10 blot show in Figure 2D
3: alpha-tubulin loading control for blots in Figure 2D
Figure_2D_700_2-Rab10_total_1.png
Top to bottom
1-3: higher contrast of Figure_2D_700_1-LRRK2_total_tubulin
2: total Rab10 blot show in Figure 2D
Figure_2D_700_3-Rab12_total_1.png
Total Rab12 shown in Figure 2C
Figure_2D_700_4-Rab12_total_2.png
Total Rab12 shown in Figure 2C, membrane incubated with the total Rab12 rabbit antibody from Proteintech
Figure_2D_800_1_LRRK2pS935.png
LRRK2 pSer935 blot shown in Figure 2D
Figure_2D_800_2_Rab10pT73.png
Rab10 pThr73 blot shown in Figure 2D
Figure_2D_800_3_Rab10_total_2.png
Total Rab10 shown in Figure 2D, membrane incubated with the rabbit anti-total Rab10 antibody from Abcam
Figure_2D_800_4-Rab12pS105.png
Phospho-Rab12 pSer105 shown in Figure 2D
Figure 3
Figure_3_700_1-LRRK2_Rab10_totals_GAPDH.png
Top to bottom
1: total LRRK2 blot shown in Figure 3. This total was multiplex with anti LRRK2 phospho-Ser935 antibody.
2: GPDH loading control for blots in Figure 3 and total Rab10 shown in Figure 3. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa.
Figure_3_700_2-Rab10_total_2.png
Total Rab10 shown in Figure 3, membrane incubated with the rabbit anti-total Rab10 antibody from Abcam.
Figure_3_700_3-LRRK2_total_for_LRRK2pS910.png
Total LRRK2 blot, not shown in the paper, multiplexed with the anti LRRK2 phospho-Ser910 antibody shown in Figure 3
Figure_3_700_4-LRRK2_totals_for_LRRK2pS955_pS973.png
Total LRRK2 blots, not shown in the paper, multiplexed with the anti LRRK2 phospho-Ser955 and phospho-Ser973 antibodies shown in Figure 3
Figure_3_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 3
2: lower contrast of Rab10 pThr73 blot shown in Figure 3
Figure_3_800_2-Rab10pT73.png
Top to bottom
1: higher contrast of LRRK2 pSer935 blot shown in Figure 3
2: Rab10 pThr73 blot shown in Figure 3
Figure_3_800_3-LRRK2pS910.png
LRRK2 pSer910 blot shown in Figure 3
Figure_3_800_4-LRRK2pS955.png
LRRK2 pSer955 blot shown in Figure 3
Figure_3_800_5-LRRK2pS973.png
LRRK2 pSer973 blot shown in Figure 3
Figure_3-quantitations.xls: raw data for charts in Figure 3
Figure 4
Figure_4A_4B_700_1-LRRK2_Rab10_totals_tubulin.png
Top to bottom
1: total LRRK2 blot shown in Figure 4A
2: total Rab10 shown in Figure 4A
3: alpha-tubulin loading control for blots in Figure 4A
4: total LRRK2 blot shown in Figure 4B
5: total Rab10 shown in Figure 4B
6: alpha-tubulin loading control for blots in Figure 4B
Figure_4A_4B_800_1-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 4A
2: Rab10 pThr73 blot shown in Figure 4A
4: LRRK2 pSer935 blot shown in Figure 4B
5: Rab10 pThr73 blot shown in Figure 4B
Figure_4A_4B-quantitations.xls: raw data for charts in Figure 4A_4B
Figure_4C_700_1-LRRK2_Rab10_totals_GAPDH_tubulin.png
Top to bottom
1: total LRRK2 blot shown in Figure 4C. This total was multiplex with anti LRRK2 phospho-Ser935 antibody.
2: GPDH loading control for blots in Figure 4C and total Rab10 shown in Figure 4C. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa
Figure_4C_800_1-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 4C
2: higher contrast of Rab10 pThr73 blot shown in Figure 4C
Figure_4C_800_2-LRRK2pS935_Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 4C
2: Rab10 pThr73 blot shown in Figure 4C
Figure_4C-quantitations.xls: raw data for charts in Figure 4C
Figure_4D-quantitations.xls: raw data for charts in Figure 4D
Figure_4D_700_1-LRRK2_total_tubulin.png
Top to bottom
1: total LRRK2 blot shown in Figure 4D
2: lower contrast of GAPDH and total Rab10 blot show in Figure 4D
3: alpha-tubulin loading control for blots in Figure 4D
Figure_4D_700_2-Rab10_total_GAPDH.png
Top to bottom
1: higher contrast of total LRRK2 blot shown in Figure 4D
2: GAPDH loading control for blots in Figure 4D and total Rab10 blot show in Figure 4D
3: higher contrast of alpha-tubulin loading control for blots in Figure 4D
Figure_4D_800_1-LRRK2pS935.png
1: LRRK2 pSer935 blot shown in Figure 4D
2: higher contrast of Rab10 pThr73 blot shown in Figure 4D
Figure_4D_800_2-Rab10pT73.png
1: lower contrast of LRRK2 pSer935 blot shown in Figure 4D
2: Rab10 pThr73 blot shown in Figure 4D
Figure 5
Figure_5A-quantitations.xls: raw data for charts in Figure 5A
Figure_5A_700_1-LRRK2_total.png
Top to bottom
1: total LRRK2 blot shown in Figure 5A
2: higher contrast of total Rab10 blot show in Figure 5A
3: higher contrast of alpha-tubulin loading control for blots in Figure 5A
Figure_5A_700_2-Rab10_total.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 5A
2: total Rab10 blot show in Figure 5A
3: higher contrast of alpha-tubulin loading control for blots in Figure 5A
Figure_5A_700_3-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 5A
2: lower contrast of total Rab10 blot show in Figure 5A
3: alpha-tubulin loading control for blots in Figure 5A
Figure_5A_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 5A
2: higher contrast of Rab10 pThr73 blot shown in Figure 5A
Figure_5A_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 5A
2: Rab10 pThr73 blot shown in Figure 5A
Figure_5B_700_1-LRRK2_total.png
Top to bottom
1: total LRRK2 blot shown in Figure 5B
2: higher contrast of total Rab10 blot show in Figure 5B
3: higher contrast of alpha-tubulin loading control for blots in Figure 5B
Figure_5B_700_2-Rab10_total.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 5B
2: total Rab10 blot show in Figure 5B
3: higher contrast of alpha-tubulin loading control for blots in Figure 5B
Figure_5B_700_3-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 5B
2: lower contrast of total Rab10 blot show in Figure 5B
3: alpha-tubulin loading control for blots in Figure 5B
Figure_5B_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 5B
2: higher contrast of Rab10 pThr73 blot shown in Figure 5B
Figure_5B_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 5B
2: Rab10 pThr73 blot shown in Figure 5B
Figure_5B-quantitations.xls: raw data for charts in Figure 5B
Figure 6
Figure_6A-quantitations.xls: raw data for charts in Figure 6A
Figure-6A_700_1-LRRK2_Rab10_totals.png
Top to bottom
1: total LRRK2 blot shown in Figure 6A
2: total Rab10 shown in Figure 6A
3: higher contrast of alpha-tubulin loading control for blots in Figure 6A
Figure_6A_700_2-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 6A
2: lower contrast of total Rab10 shown in Figure 6A
3: alpha-tubulin loading control for blots in Figure 6A
Figure_6A_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 6A
2: higher contrast of Rab10 pThr73 blot shown in Figure 6A
Figure_6A_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 6A
2: Rab10 pThr73 blot shown in Figure 6A
Figure_6B-quantitations.xls: raw data for charts in Figure 6B
Figure_6B_700_1-LRRK2_Rab10_totals.png
Top to bottom
1: total LRRK2 blot shown in Figure 6B
2: total Rab10 shown in Figure 6B
3: higher contrast of alpha-tubulin loading control for blots in Figure 6B
Figure_6B_700_2-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 6B
2: lower contrast of total Rab10 shown in Figure 6B
3: alpha-tubulin loading control for blots in Figure 6B
Figure_6B_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 6B
2: higher contrast of Rab10 pThr73 blot shown in Figure 6B
Figure_6B_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 6B
2: Rab10 pThr73 blot shown in Figure 6B
Figure_6C-quantitations.xls: raw data for charts in Figure 6C
Figure_6C_700_1-LRRK2_Rab10_totals.png
Top to bottom
1: total LRRK2 blot shown in Figure 6C
2: total Rab10 blot shown in Figure 6C
3: higher contrast of alpha-tubulin loading control for blots in Figure 6C
Figure_6C_700_2-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 6C
2: lower contrast of Rab10 shown in Figure 6C
3: alpha-tubulin loading control for blots in Figure 6C
Figure_6C_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 6C
2: higher contrast of Rab10 pThr73 blot shown in Figure 6C
Figure_6C_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 6C
2: Rab10 pThr73 blot shown in Figure 6C
Figure_6D-quantitations.xls: raw data for charts in Figure 6D
Figure_6D_700_1-LRRK2_Rab10_totals.png
Top to bottom
1: total LRRK2 blot shown in Figure 6D
2: total Rab10 blot shown in Figure 6D
3: higher contrast of alpha-tubulin loading control for blots in Figure 6D
Figure_6D_700_2-tubulin.png
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 6D
2: lower contrast of total Rab10 shown in Figure 6D
3: alpha-tubulin loading control for blots in Figure 6D
Figure_6D_800_1-LRRK2pS935.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 6D
2: higher contrast of Rab10 pThr73 blot shown in Figure 6D
Figure_6D_800_2-Rab10pT73.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 6D
2: Rab10 pThr73 blot shown in Figure 6D
Figure 7
Figure_7A-quantitations.xls: raw data for charts in Figure 7A
Figure_7A_700_1_LRRK2_total_CoIp.png
Immunoprecipitated total LRRK2 shown in Figure 7A
Figure_7A_700_2_LRRK2_total_Lysates.png (Input for the CoIp)
Top to bottom
1: total LRRK2 blot shown in Figure 7A
2: higher contrast of total Rab10 blot shown in Figure 7A
3: higher contrast of alpha-tubulin loading control for blots in Figure 7A
Figure_7A_700_3_Rab10_total_tubulin_Lysates.png (Input for the CoIp)
Top to bottom
1: lower contrast of total LRRK2 blot shown in Figure 7A
2: total Rab10 shown in Figure 7A
3: alpha-tubulin loading control for blots in Figure 7A
Figure_7A_800_1_14-3-3_CoIp.png
Blot of 14-3-3 co-immunoprecipitated with LRRK2 shown in Figure 7A
Figure_7A_800_2_LRRK2pS935_CoIp.png
Immunoprecipitated LRRK2pSer935 shown in Figure 7A
Figure_7A_800_3_LRRK2pS935_Lysates.png
Top to bottom
1: LRRK2 pSer935 blot shown in Figure 7A
2: higher contrast of Rab10 pThr73 blot shown in Figure 7A
Figure_7A_800_4_Rab10pT73_Lysates.png
Top to bottom
1: lower contrast of LRRK2 pSer935 blot shown in Figure 7A
2: Rab10 pThr73 blot shown in Figure 7A
Figure_7A_800_5_14-3-3_Lysates.png
14-3-3 blot shown in Figure 7A (Input for the CoIp)
Figure_7A_merge-700_800_LRRK2_total_LRRK2pS935.png
Merged picture (in red LRRK2 total and in green LRRK2pS935) shown in Figure 7A
Figure 7B: LSM710 laser scanning confocal microscope (Carl Zeiss) output files .lsm used to prepare Figure 7B
Figure_7B_LRRK2_HA-empty_DMSO.lsm
Figure_7B_LRRK2_HA-empty_GSK3357679A.lsm
Figure_7B_LRRK2_HA-empty_GZD-824.lsm
Figure_7B_LRRK2_HA-empty_MLi-2.lsm
Figure_7B_LRRK2_Rab29_DMSO.lsm
Figure_7B_LRRK2_ Rab29_GSK3357679A.lsm
Figure_7B_LRRK2_ Rab29_GZD-824.lsm
Figure_7B_LRRK2_ Rab29_MLi-2.lsm
Figure_7C-quantitations.xls: raw data for charts in Figure 7C
Top to bottom
1: LRRK2pSer1292 (800)
2: HA-Rab29 (700), Rab29 pThr71 (800)
3: alpha-tubulin (700) loading control for Figure 7C
4: LRRK2 total (700), LRRK2 pSer935 (800)
5: Rab10 total (700), Rab10 pThr73 (800)
6: alpha-tubulin (700) loading control for Figure 7C
Figure_7C_700_1-HA-Rab29_LRRK2_total_tubulin.png: HA-Rab29, LRRK2 total and alpha-tubulin blot loading control shown in Figure 7C
Figure_7C_700_2-Rab10_total.png: Rab10 total shown in Figure 7C
Figure_7C_800_1-LRRK2pS1292_low_contrast_Rab10pT73.png: LRRK2 pSer1292 and Rab10 pThr73 blots shown in Figure 7C
Figure_7C_800_2-LRRK2pS1292_high_contrast.png: LRRK2 pS1292 blot shown in Figure 7C
Figure_7C_800_3-LRRK2pS935.png: LRRK2 pS935 blot shown in Figure 7C
Figure_7C_800_4-Rab29pT71.png: Rab29 pThr71 blot shown in Figure 7C
Figure 8: Nikon Eclipse Ti-S fluorescence microscope output files .nd2 used to prepare Figure 8
Figure_8-quantitations-Mitophagy.xls: raw data for charts in Figure 8
Figure_8_G2019S_CTL.nd2
Figure_8_G2019S_GSK3357679A_100nM.nd2
Figure_8_G2019S_GZD-824_30nM.nd2
Figure_8_KO_CTL.nd2
Figure_8_KO_GSK3357679A_100nM.nd2
Figure_8_KO_GZD-824_30nM.nd2
Figure_8_WT_CTL.nd2
Figure_8_WT_GSK3357679A_100nM.nd2
Figure_8_WT_GZD-824_30nM.nd2
Supplementary_Figure 1_Kinase_profiling_Kinase_list.xls: code for kinase screened in Supplementary Figures 1A, 1B, 1C, 1D. Each of the enzymes has been assigned to a 2 letter code and then an abbreviation.
Supplementary_Figure 1A_Kinase_profiling_GZD-824.xls: raw data for the charts shown in Supplementary Figure 1A
Supplementary_Figure 1B_Kinase_profiling_Rebastinib.xls: raw data for the chart shown in Supplementary Figure 1B
Supplementary_Figure 1C_1D_Kinase_profiling_Ponatinib_ GSK3357679A.xls: raw data for the charts shown in Supplementary Figure 1C and 1D
The first tab on each workbook shows the layout and the position of the inhibitor in the plates used for the screen and the final assay concentration.
The inhibitors are in the same well positions throughout all the individual screen and raw data for each of the 140 kinase are reported in separated tabs. Underneath the plate layout of every kinase table is the mean for the control and blanks.
Supplementary Figure 2
Supplementary_Figure_2-700_01-LRRK2_Rab10_totals_tubulin_GAPDH.png
Top to bottom
1: total LRRK2 blot shown in Supplementary Figure 2
2: total Rab10 and GPDH loading control for blots shown in Supplementary Figure 2. Antibodies for GAPDH and Rab10 were incubated together: GAPDH is detected below 37 kDa and total Rab10 below 25 kDa.
3: alpha-tubulin loading control for blots in Supplementary Figure 2
Supplementary_Figure_2-800_01-LRRK2pS935_Rab10pT73.png
Top to bottom
1: LRRK2 pSer935 blot shown in Supplementary Figure 2
2: Rab10 pThr73 blot shown in Supplementary Figure 2
Supplementary_Figure_2-800_02- Rab10pT73_total_Abcam.png
Total Rab10 shown in Supplementary Figure 2, membrane incubated with the rabbit anti-total Rab10 antibody from Abcam
Supplementary_Figure_2-quantitations.xls: raw data for charts in Supplementary Figure 2
Table 1
Table-1_01_Ponatinib_GZD-824.xls: raw data for the IC50 values in Table 1
Table-1_02_GSK3357679A_MLi-2.xls: raw data for the IC50 values in Table 1
Table-1_03_Rebastinib.xls: raw data for the IC50 values in Table 1
Files
Figure_2A_700_1-LRRK2_Rab10_totals_GAPDH.png
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md5:a9d5e911785a58215180a6c8272279fa
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69.7 kB | Preview Download |
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md5:1c19bc8af0f8e37b2966099acaad298c
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147.3 kB | Preview Download |
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md5:929f31b84762f60cd5ddf2e7501b0118
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128.6 kB | Preview Download |
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md5:865db04312befb2c07ef9dd3f38f203b
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182.4 kB | Preview Download |
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md5:30d5d1dea4187852768c0c5a16c81c56
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75.3 kB | Preview Download |
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md5:85e55bd3c9353852469e090061dcaaed
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193.9 kB | Preview Download |
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md5:8be1d35762c2f3131ad65969386fed7b
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51.3 kB | Preview Download |
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md5:3b13ebc1c8df1dba0e646abdaecec4cd
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69.6 kB | Download |
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md5:b9024892927bcb84c176f64adf970ffe
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404.2 kB | Preview Download |
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md5:d68f3b6bbb090ccb714cdabf67141d93
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267.6 kB | Preview Download |
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md5:4a816319ddae6c627291135f8e98d679
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70.7 kB | Download |
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md5:b265fbb357bebba97e30650e9bdf9bc1
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48.6 kB | Download |
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md5:414331388eb0c8de183cb5890b6ef8fe
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180.3 kB | Preview Download |
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md5:76ed2e3b481ac9caeb6981c06586de4a
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153.6 kB | Preview Download |
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md5:8810022897d39ac185730a587ff3779e
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66.9 kB | Preview Download |
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md5:bf59711607d127199dc3bf48a0c95de8
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49.7 kB | Download |
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md5:c3f4fcc9542956a67c391f9a28b1e778
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176.8 kB | Preview Download |
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md5:e7d9405b5f45e4045c72da26a96d73dc
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178.0 kB | Preview Download |
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md5:17458e0c09109176cdddefd329e5f9d6
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150.6 kB | Preview Download |
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md5:c3f4fcc9542956a67c391f9a28b1e778
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176.8 kB | Preview Download |
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md5:a42b509e93f6f349cb9383938d5f781f
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122.1 kB | Preview Download |
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md5:3b92739c3b3ed88b656b1dec469fd332
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80.8 kB | Preview Download |
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md5:512898f4253897c7f84d742f248f32c4
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50.7 kB | Download |
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md5:a9c3e903372f9bd4d7bf48fdbe0a69e6
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625.7 kB | Preview Download |
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md5:ef9f9ff5ffd26db8243d5960f0ddb53f
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345.3 kB | Preview Download |
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md5:27ed2eebf2c0eab6ccfbd1292f110656
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657.7 kB | Preview Download |
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md5:a9c3e903372f9bd4d7bf48fdbe0a69e6
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625.7 kB | Preview Download |
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md5:ef9f9ff5ffd26db8243d5960f0ddb53f
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345.3 kB | Preview Download |
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md5:299d9b50983785fd3df9d48d5e849cb9
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873.0 kB | Preview Download |
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md5:60f2ee99c8bd6cc2613d28a2c0d7a02c
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217.2 kB | Preview Download |
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md5:949df1a8866f26d9d8adcbbc841a0b68
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846.8 kB | Preview Download |
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md5:f2d78af8f5a116ab0cc8aafe173ff02f
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291.8 kB | Preview Download |
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md5:f7a09bc950964cb79ef6fb01890d0d8e
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121.5 kB | Preview Download |
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md5:d9a668ed74c9031d73f2b86abc4b1d56
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924.7 kB | Preview Download |
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md5:1fb77ebcb6b2d4cc21d01e22495380fc
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126.6 kB | Preview Download |
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md5:7047b7ce9ada62db32f2b3a96383165d
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49.7 kB | Download |
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md5:c1667ed42c83a44fc135c73c484499c5
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40.4 kB | Download |
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md5:512b58e349f7b7fb81e9a5d27ba804da
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845.4 kB | Preview Download |
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md5:bbd0a70699143f644e8f88a285e5996d
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192.8 kB | Preview Download |
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md5:512b58e349f7b7fb81e9a5d27ba804da
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845.4 kB | Preview Download |
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md5:bbd0a70699143f644e8f88a285e5996d
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192.8 kB | Preview Download |
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md5:83a39163b1f11d015bec7eb979981a5d
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572.2 kB | Preview Download |
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md5:83a39163b1f11d015bec7eb979981a5d
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572.2 kB | Preview Download |
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md5:ed522412bc1ed09ff1cb1284fa087b99
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39.9 kB | Download |
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md5:69ab32e1973fcf1692ebf255faa8d5a0
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790.4 kB | Preview Download |
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md5:139d84979199acfa0ad308da14a6cc9a
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132.0 kB | Preview Download |
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md5:69ab32e1973fcf1692ebf255faa8d5a0
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790.4 kB | Preview Download |
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md5:139d84979199acfa0ad308da14a6cc9a
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132.0 kB | Preview Download |
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md5:a1e03fdc9da9b60b05928fbf10fb52e0
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1.2 MB | Preview Download |
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md5:c1bde4ce0b13831ccd0e2af5a7cabb7b
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491.7 kB | Preview Download |
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md5:509ea2267a0ea2153a360f7dfa0e2b61
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42.5 kB | Download |
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md5:f7c18e9c9623aa6fc8223700ce01df9c
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687.7 kB | Preview Download |
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md5:ff411452534ca363c6d49a87e883a633
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650.7 kB | Preview Download |
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md5:0bf2985a103a9a09c38763e7d3d88d05
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458.9 kB | Preview Download |
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md5:f7c18e9c9623aa6fc8223700ce01df9c
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687.7 kB | Preview Download |
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md5:0bf2985a103a9a09c38763e7d3d88d05
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458.9 kB | Preview Download |
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md5:2f1b0c67971532def236a0574a8403b7
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1.0 MB | Preview Download |
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md5:a292eddab53da95269d9b70ff24b6205
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576.8 kB | Preview Download |
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md5:31f4e4ff4e5490a23b6c940a882fa5d9
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42.5 kB | Download |
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md5:27969dd93133c962e65d16f71ec22b14
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744.8 kB | Preview Download |
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md5:4536e852e1356e9a377fb69110e93cb4
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677.2 kB | Preview Download |
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md5:06be18e560d0fa4cc8502c3a954b7cb4
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489.9 kB | Preview Download |
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md5:27969dd93133c962e65d16f71ec22b14
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744.8 kB | Preview Download |
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md5:06be18e560d0fa4cc8502c3a954b7cb4
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489.9 kB | Preview Download |
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md5:d0f23884194c7df662de740c414fb24c
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711.9 kB | Preview Download |
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md5:d0f23884194c7df662de740c414fb24c
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711.9 kB | Preview Download |
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md5:2e3a13803aed97bf5afb27c6f47c94e4
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34.8 kB | Download |
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md5:1f425fb931656a2f98b009ebb40beb5f
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10.4 kB | Preview Download |
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md5:5aab205d270ca82c33a1e84436b49478
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69.5 kB | Preview Download |
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md5:5511b830c88d216f262feb9a624023c2
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114.8 kB | Preview Download |
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md5:1f425fb931656a2f98b009ebb40beb5f
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10.4 kB | Preview Download |
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md5:5aab205d270ca82c33a1e84436b49478
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69.5 kB | Preview Download |
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md5:5511b830c88d216f262feb9a624023c2
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114.8 kB | Preview Download |
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md5:77e65e9913c214c72818f9cc7d58166e
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35.8 kB | Preview Download |
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md5:0663a0c9fd2890102ea215c2a5f7c515
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9.5 kB | Preview Download |
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md5:5e038e9c5163b2eb7e4dd6bee8d1c41f
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9.3 kB | Preview Download |
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md5:3dc3a63348c02d4358ac72f1fd8f4175
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98.5 kB | Preview Download |
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md5:5b93962dda5591b58cda848961861b4f
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129.4 kB | Preview Download |
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md5:7a162a8313f8372552af8baa3ee6a5b5
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16.6 kB | Preview Download |
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md5:7c64f47685735a1c807df5da553bb1f8
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22.1 MB | Download |
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md5:a0766cd12d726116392cbd3232bd2db4
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22.1 MB | Download |
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md5:3a55b1764fb45c7aef57e5ac67b67919
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22.1 MB | Download |
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md5:42989e43bd6e521ad5173db8160fc76d
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22.1 MB | Download |
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md5:eae034cf3e976cf72334900a4ed164ee
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22.1 MB | Download |
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md5:387c4ee5021c6d08306521d906ac959e
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22.1 MB | Download |
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md5:ebb79bf6b47e14d6bc034badaba71a8a
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22.1 MB | Download |
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md5:94cf4758adddbfc93e55d23c7d1d729c
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22.1 MB | Download |
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md5:28e472a094ad9a870f0d1b9ddad8eed4
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56.3 kB | Download |
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md5:6b512d0fc18d217e1faf8f46151b0bac
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85.0 kB | Preview Download |
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md5:6f26f4c9a6941fd28feb56104c907587
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390.2 kB | Preview Download |
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md5:6b512d0fc18d217e1faf8f46151b0bac
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85.0 kB | Preview Download |
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md5:6f26f4c9a6941fd28feb56104c907587
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390.2 kB | Preview Download |
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md5:207c80df120796257ee4540c764ac0bc
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337.2 kB | Preview Download |
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md5:207c80df120796257ee4540c764ac0bc
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337.2 kB | Preview Download |
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md5:e13d72b9b6e8d18c84b027dcd2fd22f7
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513.7 kB | Preview Download |
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md5:e13d72b9b6e8d18c84b027dcd2fd22f7
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513.7 kB | Preview Download |
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md5:9461040e44d49a15ff9709bbdfa4ece2
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25.9 kB | Preview Download |
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md5:03f58d025c2cbced828fb67318983320
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40.7 kB | Preview Download |
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md5:5da5d5cbba5faa9fe7cf6d63ec751859
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11.3 kB | Download |
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md5:5c11bab8ff25245505a15e15032386c7
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11.7 MB | Download |
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md5:2c0557654871220f3d78ce81139019ea
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11.7 MB | Download |
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md5:37b05b32a828fa0c6f8ad7c5aa1c2925
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11.7 MB | Download |
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md5:16965975dcc963ba030ce0d29da4a926
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11.7 MB | Download |
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md5:8f772f080dfd5e73c31e2065a426fc79
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11.7 MB | Download |
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md5:3b2624309216457a6b9fd078d980764e
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11.7 MB | Download |
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md5:236c09ee8bdc1dc4cc2ce515b8081379
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11.7 MB | Download |
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md5:3e72013a79114d21c68e16f1e27390a4
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11.7 MB | Download |
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md5:8e252fc4eb1354e092b963e7d910d082
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11.7 MB | Download |
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md5:8e252fc4eb1354e092b963e7d910d082
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11.7 MB | Download |
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md5:f798b61c4389f0fbf0e5a8e280eda46c
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14.9 kB | Download |
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md5:dd7e80a0e8e5cbbd8465e2974bdd2a11
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4.1 MB | Download |
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md5:e4aff9b38bb6631222676d081fbe4a8f
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4.0 MB | Download |
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md5:3562272c25f482a5a02fe86fadc508a4
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3.5 MB | Download |
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md5:d584094c85d32d78c1c9c7679515aadc
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93.0 kB | Preview Download |
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md5:d584094c85d32d78c1c9c7679515aadc
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93.0 kB | Preview Download |
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md5:d7085a8ee0f222dbdc58e2b5b33d3d23
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58.1 kB | Preview Download |
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md5:c40673a995a2eb38b162cac937661911
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29.5 kB | Preview Download |
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md5:ce9e3ff553142b6fc0509fcc7c4f0349
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36.9 kB | Download |
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md5:5a044eda2d6b409d5efe0b556ae2a0c8
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46.1 kB | Download |
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md5:536ae7f38437de9ea26fa5f8b000f403
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38.9 kB | Download |
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md5:df851a82e041204b6811d40257cb7e85
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51.0 kB | Download |
