Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation
- Publication Date
- August 28, 2012
- Journal
- PLOS ONE
- Authors
- Spencer B. Hermanson, Coby B. Carlson, Steven M. Riddle, Jing Zhao, et al
- Volume
- 7
- Issue
- 8
- Pages
- e43580
- DOI
- https://dx.plos.org/10.1371/journal.pone.0043580
- Publisher URL
- http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0043580
- PubMed
- http://www.ncbi.nlm.nih.gov/pubmed/22952710
- PubMed Central
- http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429506
- Europe PMC
- http://europepmc.org/abstract/MED/22952710
- Web of Science
- 000308213600032
- Scopus
- 84865515027
- Mendeley
- http://www.mendeley.com/research/screening-novel-lrrk2-inhibitors-using-highthroughput-trfret-cellular-assay-lrrk2-ser935-phosphoryla
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Mendeley | Further Information
{"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}CrossRef
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Relative Metric
{"start_date"=>"2012-01-01T00:00:00Z", "end_date"=>"2012-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[322, 550, 671, 773, 864, 955, 1048, 1135, 1223, 1308, 1387, 1465, 1534, 1602, 1673, 1744, 1813, 1885, 1955, 2026, 2093, 2160, 2228, 2290, 2349]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[316, 541, 663, 766, 856, 950, 1041, 1128, 1218, 1302, 1382, 1456, 1526, 1593, 1657, 1729, 1796, 1862, 1930, 1999, 2065, 2132, 2202, 2261, 2319]}, {"subject_area"=>"/Biology and life sciences/Molecular biology", "average_usage"=>[324, 551, 679, 782, 879, 971, 1064, 1154, 1245, 1328, 1406, 1476, 1541, 1617, 1683, 1754, 1818, 1893, 1962, 2025, 2092, 2164, 2227, 2294, 2355]}, {"subject_area"=>"/Biology and life sciences/Toxicology", "average_usage"=>[320, 550, 676, 777, 874, 964, 1079, 1158, 1235, 1357, 1433, 1500, 1567, 1635, 1707, 1802, 1875, 1938, 1987, 2062, 2140, 2206, 2273, 2328, 2385]}, {"subject_area"=>"/Medicine and health sciences/Pathology and laboratory medicine", "average_usage"=>[321, 553, 683, 785, 880, 973, 1072, 1162, 1249, 1336, 1425, 1504, 1574, 1642, 1711, 1785, 1852, 1923, 1987, 2050, 2129, 2192, 2259, 2325, 2393]}]}Loading … 

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