Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer
Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throu...
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| Veröffentlicht in: | Biological & pharmaceutical bulletin 2017/09/01, Vol.40(9), pp.1454-1462 |
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| creator | Kim, Tae Gi Lee, Ju Hee Lee, Mi Young Kim, Ka-Ul Lee, Jeong Hyun Park, Chi Hoon Lee, Byung Ho Oh, Kwang-Seok |
| description | Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z′ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z′ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries. |
| doi_str_mv | 10.1248/bpb.b17-00283 |
| format | Article |
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In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z′ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z′ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.</description><identifier>ISSN: 0918-6158</identifier><identifier>EISSN: 1347-5215</identifier><identifier>DOI: 10.1248/bpb.b17-00283</identifier><identifier>PMID: 28867728</identifier><language>eng</language><publisher>Japan: The Pharmaceutical Society of Japan</publisher><subject>Assaying ; Biotin - chemistry ; Cell Cycle Proteins - antagonists & inhibitors ; Cell proliferation ; Cell Proliferation - drug effects ; Dose-Response Relationship, Drug ; Energy transfer ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Fluorescence resonance energy transfer ; Fluorescence Resonance Energy Transfer - methods ; HeLa Cells ; high-throughput screening ; High-Throughput Screening Assays - methods ; Humans ; Inhibitors ; Iodides ; Menadione ; polo-box domain ; Polo-like kinase ; Polo-Like Kinase 1 ; Protein Binding - drug effects ; Protein Serine-Threonine Kinases - antagonists & inhibitors ; protein–protein interaction ; Proto-Oncogene Proteins - antagonists & inhibitors ; Reproducibility of Results ; Small Molecule Libraries ; Structure-Activity Relationship</subject><ispartof>Biological and Pharmaceutical Bulletin, 2017/09/01, Vol.40(9), pp.1454-1462</ispartof><rights>2017 The Pharmaceutical Society of Japan</rights><rights>Copyright Japan Science and Technology Agency 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c702t-d72ddcad4a59e26380e41514c2a46678fe2ad54f38f1c37f9fe5ae8a67ce88283</citedby><cites>FETCH-LOGICAL-c702t-d72ddcad4a59e26380e41514c2a46678fe2ad54f38f1c37f9fe5ae8a67ce88283</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,1883,4024,27923,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28867728$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, Tae Gi</creatorcontrib><creatorcontrib>Lee, Ju Hee</creatorcontrib><creatorcontrib>Lee, Mi Young</creatorcontrib><creatorcontrib>Kim, Ka-Ul</creatorcontrib><creatorcontrib>Lee, Jeong Hyun</creatorcontrib><creatorcontrib>Park, Chi Hoon</creatorcontrib><creatorcontrib>Lee, Byung Ho</creatorcontrib><creatorcontrib>Oh, Kwang-Seok</creatorcontrib><creatorcontrib>bGraduate School of New Drug Discovery and Development</creatorcontrib><creatorcontrib>Korea Research Institute of Chemical Technology</creatorcontrib><creatorcontrib>cDepartment of Medicinal and Pharmaceutical Chemistry</creatorcontrib><creatorcontrib>University of Science and Technology</creatorcontrib><creatorcontrib>Chungnam National University</creatorcontrib><creatorcontrib>aBio-Organic Science Division</creatorcontrib><title>Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer</title><title>Biological & pharmaceutical bulletin</title><addtitle>Biol Pharm Bull</addtitle><description>Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z′ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z′ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.</description><subject>Assaying</subject><subject>Biotin - chemistry</subject><subject>Cell Cycle Proteins - antagonists & inhibitors</subject><subject>Cell proliferation</subject><subject>Cell Proliferation - drug effects</subject><subject>Dose-Response Relationship, Drug</subject><subject>Energy transfer</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Fluorescence</subject><subject>Fluorescence resonance energy transfer</subject><subject>Fluorescence Resonance Energy Transfer - methods</subject><subject>HeLa Cells</subject><subject>high-throughput screening</subject><subject>High-Throughput Screening Assays - methods</subject><subject>Humans</subject><subject>Inhibitors</subject><subject>Iodides</subject><subject>Menadione</subject><subject>polo-box domain</subject><subject>Polo-like kinase</subject><subject>Polo-Like Kinase 1</subject><subject>Protein Binding - drug effects</subject><subject>Protein Serine-Threonine Kinases - antagonists & inhibitors</subject><subject>protein–protein interaction</subject><subject>Proto-Oncogene Proteins - antagonists & inhibitors</subject><subject>Reproducibility of Results</subject><subject>Small Molecule Libraries</subject><subject>Structure-Activity Relationship</subject><issn>0918-6158</issn><issn>1347-5215</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkU1v1DAQhiMEotvCkSuyxDmt7dixc2y3n2IlEFrOluNMNl4SO9hJ1f0j_F68u2W5zEgzj975eLPsE8GXhDJ5VY_1ZU1EjjGVxZtsQQomck4Jf5stcEVkXhIuz7LzGLcYY4Fp8T47o1KWQlC5yP7cwjP0fhzATci3SKNHu-nydRf8vOnGeULXMeodan1AT66ztZ18iHty6gB9973Pb_wLuvWDtm5fPpRW9hegr9bpCIigm5Qa5B1a2wHyHxB9_5wK9_3sA0QDzgC6cxA2O7QO2sUWwofsXav7CB9f80X28_5uvXzMV98enpbXq9ykS6a8EbRpjG6Y5hXQspAYGOGEGapZWQrZAtUNZ20hW2IK0VYtcA1Sl8KAlOlhF9mXo-4Y_O8Z4qS2fg4ujVSkKngpSClwovIjZYKPMUCrxmAHHXaKYLV3QSUXVHJBHVxI_OdX1bkeoDnR_96egIcjkLrW6N673jr4P9tEUdv0SEXxQZRhXClMSkUYZymUlFacc1YlpeVRaRsnvYHTKB0ma3o4LMawqvbhtOCpazodFLjiL3Bmsks</recordid><startdate>2017</startdate><enddate>2017</enddate><creator>Kim, Tae Gi</creator><creator>Lee, Ju Hee</creator><creator>Lee, Mi Young</creator><creator>Kim, Ka-Ul</creator><creator>Lee, Jeong Hyun</creator><creator>Park, Chi Hoon</creator><creator>Lee, Byung Ho</creator><creator>Oh, Kwang-Seok</creator><general>The Pharmaceutical Society of Japan</general><general>Pharmaceutical Society of Japan</general><general>Japan Science and Technology Agency</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope></search><sort><creationdate>2017</creationdate><title>Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer</title><author>Kim, Tae Gi ; 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In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z′ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z′ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.</abstract><cop>Japan</cop><pub>The Pharmaceutical Society of Japan</pub><pmid>28867728</pmid><doi>10.1248/bpb.b17-00283</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| source | J-STAGE Free; MEDLINE; EZB-FREE-00999 freely available EZB journals; Free Full-Text Journals in Chemistry |
| subjects | Assaying Biotin - chemistry Cell Cycle Proteins - antagonists & inhibitors Cell proliferation Cell Proliferation - drug effects Dose-Response Relationship, Drug Energy transfer Enzyme-Linked Immunosorbent Assay Fluorescence Fluorescence resonance energy transfer Fluorescence Resonance Energy Transfer - methods HeLa Cells high-throughput screening High-Throughput Screening Assays - methods Humans Inhibitors Iodides Menadione polo-box domain Polo-like kinase Polo-Like Kinase 1 Protein Binding - drug effects Protein Serine-Threonine Kinases - antagonists & inhibitors protein–protein interaction Proto-Oncogene Proteins - antagonists & inhibitors Reproducibility of Results Small Molecule Libraries Structure-Activity Relationship |
| title | Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer |
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