Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation
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{"title"=>"Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation", "type"=>"journal", "authors"=>[{"first_name"=>"Spencer B.", "last_name"=>"Hermanson", "scopus_author_id"=>"55355517200"}, {"first_name"=>"Coby B.", "last_name"=>"Carlson", "scopus_author_id"=>"7202968650"}, {"first_name"=>"Steven M.", "last_name"=>"Riddle", "scopus_author_id"=>"14066320400"}, {"first_name"=>"Jing", "last_name"=>"Zhao", "scopus_author_id"=>"57191637510"}, {"first_name"=>"Kurt W.", "last_name"=>"Vogel", "scopus_author_id"=>"8346162900"}, {"first_name"=>"R. Jeremy", "last_name"=>"Nichols", "scopus_author_id"=>"16833969400"}, {"first_name"=>"Kun", "last_name"=>"Bi", "scopus_author_id"=>"15848004400"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84865515027", "doi"=>"10.1371/journal.pone.0043580", "pui"=>"365529052", "issn"=>"19326203", "pmid"=>"22952710", "scopus"=>"2-s2.0-84865515027"}, "id"=>"76d7a9eb-37d6-37e9-86f9-8acc3beab4e3", "abstract"=>"BACKGROUND: Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.\\n\\nCONCLUSIONS/SIGNIFICANCE: We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.", "link"=>"http://www.mendeley.com/research/screening-novel-lrrk2-inhibitors-using-highthroughput-trfret-cellular-assay-lrrk2-ser935-phosphoryla", "reader_count"=>37, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>15, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>5, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>5, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>15, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>5, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>5, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>9, "Neuroscience"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>2, "Chemistry"=>5, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>9}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>5}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>2}}, "reader_count_by_country"=>{"United States"=>1, "Egypt"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/306966", "https://ndownloader.figshare.com/files/306995", "https://ndownloader.figshare.com/files/307032"], "description"=>"<div><h3>Background</h3><p>Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.</p> <h3>Methodology/Principal Findings</h3><p>Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.</p> <h3>Conclusions/Significance</h3><p>We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity <em>in vitro</em>.</p> </div>", "links"=>[], "tags"=>["lrrk2", "inhibitors", "high-throughput", "tr-fret", "cellular", "assay", "ser935", "phosphorylation"], "article_id"=>120556, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0043580.s001", "https://dx.doi.org/10.1371/journal.pone.0043580.s002", "https://dx.doi.org/10.1371/journal.pone.0043580.s003"], "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Screening_for_Novel_LRRK2_Inhibitors_Using_a_High_Throughput_TR_FRET_Cellular_Assay_for_LRRK2_Ser935_Phosphorylation/120556", "title"=>"Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-08-28 00:09:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/585332"], "description"=>"<p>U-2 OS cells were transduced with 20% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. (A) GFP images were captured and representative images are shown. (B) Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody. The quantifications are from 3 independent experiments.</p>", "links"=>[], "tags"=>["lrrk2-gfp", "u-2", "os", "cells", "bacmam"], "article_id"=>255815, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_LRRK2_GFP_in_U_2_OS_cells_via_BacMam_gene_delivery_/255815", "title"=>"Expression of LRRK2-GFP in U-2 OS cells via BacMam gene delivery.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:36:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/585684"], "description"=>"<p>Average percent inhibition of library compounds was analyzed to produce a frequency distribution with binning equal to one percent increments. The frequency distribution was plotted and a gaussian curve fit to the distribution (curve statistics: mean 4.38% standard, deviation 10.53% and R2 value of 0.962).</p>", "links"=>[], "tags"=>["mini"], "article_id"=>256181, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Tocriscreen_8482_mini_library_screening_results_/256181", "title"=>"Tocriscreen™ mini library screening results.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:43:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/585752"], "description"=>"<p>Flp-In T-REx™ HEK293 GFP-LRRK2 G2019S cells (GS) or A2016T/G2019S (IRM) were induced and treated with indicated compounds at 20 µM. After cell lysis, GFP-LRRK2 was immunoprecipitated and the phosphorylation of LRRK2 was analyzed by immunoblotting. The quantifications are from 3 independent experiments.</p>", "links"=>[], "tags"=>["compounds", "lrrk2", "phosphorylation", "serines", "955"], "article_id"=>256241, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g006", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effects_of_hit_compounds_on_LRRK2_phosphorylation_at_serines_910_935_955_and_973_/256241", "title"=>"The effects of hit compounds on LRRK2 phosphorylation at serines 910, 935, 955 and 973.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:44:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/585450"], "description"=>"<p>(A) U-2 OS cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were plated onto a 384-well assay plate and lysed in the presence of Tb-labeled anti-pSer935 antibody. TR-FRET was measured and the emission ratios of 520 nm/490 nm are plotted against the amount of BacMam used for transduction. (B) Cells transduced with 20% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (A). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.</p>", "links"=>[], "tags"=>["detection", "ser935", "phosphorylation", "lrrk2-gfp", "u-2", "os"], "article_id"=>255933, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g002", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TR_FRET_detection_of_Ser935_phosphorylation_of_LRRK2_GFP_in_U_2_OS_cells_/255933", "title"=>"TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in U-2 OS cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:38:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/585891"], "description"=>"<p>IC<sub>50</sub> (µM) +/− SD; *Partial inhibition; nd: not tested.</p>", "links"=>[], "tags"=>["compounds", "tr-fret", "cell-based", "assay", "ser935", "phosphorylation"], "article_id"=>256377, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.t001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_IC_50_values_181_M_of_known_compounds_determined_in_the_TR_FRET_cell_based_assay_for_Ser935_phosphorylation_of_LRRK2_/256377", "title"=>"IC<sub>50</sub> values (µM) of known compounds determined in the TR-FRET cell-based assay for Ser935 phosphorylation of LRRK2.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-28 01:46:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/585927"], "description"=>"<p>The IC<sub>50</sub> values are the averaged numbers from three independent experiments for both cellular and biochemical TR-FRET assays and one representative data of two independent experiments for the cytotoxicity assay.</p>", "links"=>[], "tags"=>["16", "hits", "mini"], "article_id"=>256421, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.t002", "stats"=>{"downloads"=>4, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Top_16_hits_of_the_Tocriscreen_8482_Mini_library_screen_/256421", "title"=>"Top 16 hits of the Tocriscreen™ Mini library screen.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-28 01:47:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/585631"], "description"=>"<p>Human neural stem cells were transduced with 10% BacMam LRRK2-GFP G2019S or D1994A. Cells were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043580#pone-0043580-g002\" target=\"_blank\">Figure 2</a>. Emission ratios of 520 nm/495 nm are plotted against the concentration of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.</p>", "links"=>[], "tags"=>["detection", "ser935", "phosphorylation", "lrrk2-gfp", "neural"], "article_id"=>256120, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g004", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TR_FRET_detection_of_Ser935_phosphorylation_of_LRRK2_GFP_in_human_neural_stem_cells_/256120", "title"=>"TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in human neural stem cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:42:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/585522"], "description"=>"<p>(A) SH-SY5Y cells were transduced with 25% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody was performed. The quantifications are from 3 independent experiments. (B) SH-SY5Y cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C and D1994A. Cells were plated onto a 384-well assay plate, lysed and analyzed as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043580#pone-0043580-g002\" target=\"_blank\">Figure 2</a> legend. Emission ratios of 520 nm/495 nm are plotted against the amount of BacMam used for transduction. (C) Cells transduced with 25% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (B). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.</p>", "links"=>[], "tags"=>["detection", "ser935", "phosphorylation", "lrrk2-gfp", "sh-sy5y"], "article_id"=>256005, "categories"=>["Biotechnology", "Neuroscience"], "users"=>["Spencer B. Hermanson", "Coby B. Carlson", "Steven M. Riddle", "Jing Zhao", "Kurt W. Vogel", "R. Jeremy Nichols", "Kun Bi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043580.g003", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TR_FRET_detection_of_Ser935_phosphorylation_of_LRRK2_GFP_in_SH_SY5Y_cells_/256005", "title"=>"TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in SH-SY5Y cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:40:05"}

PMC Usage Stats | Further Information

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Relative Metric

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