Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators
The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding spec...
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| Veröffentlicht in: | PloS one 2013, Vol.8 (10), p.e78042-e78042 |
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| creator | Xie, Xingliang Sun, Lin Pessetto, Ziyan Yuan Zhao, Yan Zang, Zhihe Zhong, Ling Wu, Min Su, Qing Gao, Xiurong Zan, Wang Sun, Yiyi |
| description | The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction. |
| doi_str_mv | 10.1371/journal.pone.0078042 |
| format | Article |
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It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0078042</identifier><identifier>PMID: 24205080</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Assaying ; Binding ; Cbl protein ; Cell cycle ; Diabetes ; Epidermal growth factor ; Fluorescence ; Fluorescence polarization ; Fluorescence Polarization - methods ; Inhibition ; Interfaces ; Kinases ; Libraries ; Lymphoma ; Lymphoma - enzymology ; Lymphomas ; Medical screening ; Peptides ; Pharmacy ; Polarization ; Protein Binding ; Protein interaction ; Proteins ; Proto-Oncogene Proteins c-cbl - metabolism ; Regulators ; Rodents ; Screens ; Signal transduction ; Transduction ; Ubiquitin ; Ubiquitin-Conjugating Enzymes - metabolism ; Ubiquitin-protein ligase</subject><ispartof>PloS one, 2013, Vol.8 (10), p.e78042-e78042</ispartof><rights>2013 Xie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Xie et al 2013 Xie et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-2858582b5b832ccdeec1622c9d68e4b9d3230defbcde0682b09f806a670623263</citedby><cites>FETCH-LOGICAL-c526t-2858582b5b832ccdeec1622c9d68e4b9d3230defbcde0682b09f806a670623263</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814989/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814989/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,4010,23845,27900,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24205080$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Xie, Xingliang</creatorcontrib><creatorcontrib>Sun, Lin</creatorcontrib><creatorcontrib>Pessetto, Ziyan Yuan</creatorcontrib><creatorcontrib>Zhao, Yan</creatorcontrib><creatorcontrib>Zang, Zhihe</creatorcontrib><creatorcontrib>Zhong, Ling</creatorcontrib><creatorcontrib>Wu, Min</creatorcontrib><creatorcontrib>Su, Qing</creatorcontrib><creatorcontrib>Gao, Xiurong</creatorcontrib><creatorcontrib>Zan, Wang</creatorcontrib><creatorcontrib>Sun, Yiyi</creatorcontrib><title>Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.</description><subject>Assaying</subject><subject>Binding</subject><subject>Cbl protein</subject><subject>Cell cycle</subject><subject>Diabetes</subject><subject>Epidermal growth factor</subject><subject>Fluorescence</subject><subject>Fluorescence polarization</subject><subject>Fluorescence Polarization - methods</subject><subject>Inhibition</subject><subject>Interfaces</subject><subject>Kinases</subject><subject>Libraries</subject><subject>Lymphoma</subject><subject>Lymphoma - enzymology</subject><subject>Lymphomas</subject><subject>Medical screening</subject><subject>Peptides</subject><subject>Pharmacy</subject><subject>Polarization</subject><subject>Protein Binding</subject><subject>Protein interaction</subject><subject>Proteins</subject><subject>Proto-Oncogene Proteins c-cbl - metabolism</subject><subject>Regulators</subject><subject>Rodents</subject><subject>Screens</subject><subject>Signal transduction</subject><subject>Transduction</subject><subject>Ubiquitin</subject><subject>Ubiquitin-Conjugating Enzymes - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xie, Xingliang</au><au>Sun, Lin</au><au>Pessetto, Ziyan Yuan</au><au>Zhao, Yan</au><au>Zang, Zhihe</au><au>Zhong, Ling</au><au>Wu, Min</au><au>Su, Qing</au><au>Gao, Xiurong</au><au>Zan, Wang</au><au>Sun, Yiyi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013</date><risdate>2013</risdate><volume>8</volume><issue>10</issue><spage>e78042</spage><epage>e78042</epage><pages>e78042-e78042</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24205080</pmid><doi>10.1371/journal.pone.0078042</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Assaying Binding Cbl protein Cell cycle Diabetes Epidermal growth factor Fluorescence Fluorescence polarization Fluorescence Polarization - methods Inhibition Interfaces Kinases Libraries Lymphoma Lymphoma - enzymology Lymphomas Medical screening Peptides Pharmacy Polarization Protein Binding Protein interaction Proteins Proto-Oncogene Proteins c-cbl - metabolism Regulators Rodents Screens Signal transduction Transduction Ubiquitin Ubiquitin-Conjugating Enzymes - metabolism Ubiquitin-protein ligase |
| title | Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators |
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