Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90
High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment....
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description | High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis. |
doi_str_mv | 10.1089/adt.2005.3.273 |
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These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.</description><identifier>ISSN: 1540-658X</identifier><identifier>EISSN: 1557-8127</identifier><identifier>DOI: 10.1089/adt.2005.3.273</identifier><identifier>PMID: 15971989</identifier><language>eng</language><publisher>United States</publisher><subject><![CDATA[Animals ; Benzoquinones ; Blotting, Western ; Carrier Proteins - antagonists & inhibitors ; Carrier Proteins - metabolism ; Cell Extracts - analysis ; Dose-Response Relationship, Drug ; Drug Design ; eIF-2 Kinase - antagonists & inhibitors ; eIF-2 Kinase - metabolism ; Enzyme Inhibitors - pharmacology ; HCT116 Cells ; Heterocyclic Compounds, 2-Ring - pharmacology ; HSP70 Heat-Shock Proteins - antagonists & inhibitors ; HSP70 Heat-Shock Proteins - metabolism ; HSP90 Heat-Shock Proteins - antagonists & inhibitors ; HSP90 Heat-Shock Proteins - metabolism ; HT29 Cells ; Humans ; Immunoassay - methods ; Lactams, Macrocyclic ; Lymphocytes - chemistry ; Lymphocytes - drug effects ; Lymphocytes - metabolism ; Mice ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-raf - antagonists & inhibitors ; Proto-Oncogene Proteins c-raf - metabolism ; Pyrazoles - pharmacology ; Quinones - pharmacology ; Rifabutin - analogs & derivatives ; Rifabutin - pharmacology ; Technology, Pharmaceutical - methods ; Technology, Pharmaceutical - trends ; Transplantation, Heterologous]]></subject><ispartof>Assay and drug development technologies, 2005-06, Vol.3 (3), p.273-285</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c324t-7905b2c4bb6fb09e21d36aeddbece3efbc58135d369f8af14e6f31ac59d5e6183</citedby><cites>FETCH-LOGICAL-c324t-7905b2c4bb6fb09e21d36aeddbece3efbc58135d369f8af14e6f31ac59d5e6183</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,3029,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15971989$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hardcastle, Anthea</creatorcontrib><creatorcontrib>Boxall, Kathy</creatorcontrib><creatorcontrib>Richards, Juliet</creatorcontrib><creatorcontrib>Tomlin, Peter</creatorcontrib><creatorcontrib>Sharp, Swee</creatorcontrib><creatorcontrib>Clarke, Paul</creatorcontrib><creatorcontrib>Workman, Paul</creatorcontrib><creatorcontrib>Aherne, Wynne</creatorcontrib><title>Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90</title><title>Assay and drug development technologies</title><addtitle>Assay Drug Dev Technol</addtitle><description>High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.</description><subject>Animals</subject><subject>Benzoquinones</subject><subject>Blotting, Western</subject><subject>Carrier Proteins - antagonists & inhibitors</subject><subject>Carrier Proteins - metabolism</subject><subject>Cell Extracts - analysis</subject><subject>Dose-Response Relationship, Drug</subject><subject>Drug Design</subject><subject>eIF-2 Kinase - antagonists & inhibitors</subject><subject>eIF-2 Kinase - metabolism</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>HCT116 Cells</subject><subject>Heterocyclic Compounds, 2-Ring - pharmacology</subject><subject>HSP70 Heat-Shock Proteins - antagonists & inhibitors</subject><subject>HSP70 Heat-Shock Proteins - metabolism</subject><subject>HSP90 Heat-Shock Proteins - antagonists & inhibitors</subject><subject>HSP90 Heat-Shock Proteins - metabolism</subject><subject>HT29 Cells</subject><subject>Humans</subject><subject>Immunoassay - methods</subject><subject>Lactams, Macrocyclic</subject><subject>Lymphocytes - chemistry</subject><subject>Lymphocytes - drug effects</subject><subject>Lymphocytes - metabolism</subject><subject>Mice</subject><subject>Neoplasm Transplantation</subject><subject>Proto-Oncogene Proteins c-raf - antagonists & inhibitors</subject><subject>Proto-Oncogene Proteins c-raf - metabolism</subject><subject>Pyrazoles - pharmacology</subject><subject>Quinones - pharmacology</subject><subject>Rifabutin - analogs & derivatives</subject><subject>Rifabutin - pharmacology</subject><subject>Technology, Pharmaceutical - methods</subject><subject>Technology, Pharmaceutical - trends</subject><subject>Transplantation, Heterologous</subject><issn>1540-658X</issn><issn>1557-8127</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkctq3jAQhUVpaS7tNsuiVXd2JMuyre5CSJNAoIu20J3QZVwrtSxXkgP_0_RVK5MfssxKozPfnGE4CF1QUlMyiEtlc90QwmtWNz17g04p53010KZ_u9ctqTo-_DpBZyk9EtIQ1rfv0QnloqdiEKfo3_cwO1utk0qAnffbElRK6pCwW7AHM6nFJV_p0rbYxu03ti6Z8ATx8AXnCVzEal1nZ1R2YdmHiogtPMEcVg9LxmEs6uS0yyGm_bcDPsxgtllFXDasEMMCeAKVqzQF8wevMWQoXoJ8QO9GNSf4eHzP0c-vNz-u76qHb7f311cPlWFNm6teEK4b02rdjZoIaKhlnQJrNRhgMGrDB8p4EcU4qJG20I2MKsOF5dDRgZ2jz8--ZfXfDVKWvtwJ86wWCFuSXS86ItrXQdrztqOCFbB-Bk0MKUUY5RqdV_EgKZF7drJkJ_fsJJMluzLw6ei8aQ_2BT-Gxf4D1f6aEQ</recordid><startdate>200506</startdate><enddate>200506</enddate><creator>Hardcastle, Anthea</creator><creator>Boxall, Kathy</creator><creator>Richards, Juliet</creator><creator>Tomlin, Peter</creator><creator>Sharp, Swee</creator><creator>Clarke, Paul</creator><creator>Workman, Paul</creator><creator>Aherne, Wynne</creator><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>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>200506</creationdate><title>Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90</title><author>Hardcastle, Anthea ; Boxall, Kathy ; Richards, Juliet ; Tomlin, Peter ; Sharp, Swee ; Clarke, Paul ; Workman, Paul ; Aherne, Wynne</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c324t-7905b2c4bb6fb09e21d36aeddbece3efbc58135d369f8af14e6f31ac59d5e6183</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Animals</topic><topic>Benzoquinones</topic><topic>Blotting, Western</topic><topic>Carrier Proteins - antagonists & inhibitors</topic><topic>Carrier Proteins - metabolism</topic><topic>Cell Extracts - analysis</topic><topic>Dose-Response Relationship, Drug</topic><topic>Drug Design</topic><topic>eIF-2 Kinase - antagonists & inhibitors</topic><topic>eIF-2 Kinase - metabolism</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>HCT116 Cells</topic><topic>Heterocyclic Compounds, 2-Ring - pharmacology</topic><topic>HSP70 Heat-Shock Proteins - antagonists & inhibitors</topic><topic>HSP70 Heat-Shock Proteins - metabolism</topic><topic>HSP90 Heat-Shock Proteins - antagonists & inhibitors</topic><topic>HSP90 Heat-Shock Proteins - metabolism</topic><topic>HT29 Cells</topic><topic>Humans</topic><topic>Immunoassay - methods</topic><topic>Lactams, Macrocyclic</topic><topic>Lymphocytes - chemistry</topic><topic>Lymphocytes - drug effects</topic><topic>Lymphocytes - metabolism</topic><topic>Mice</topic><topic>Neoplasm Transplantation</topic><topic>Proto-Oncogene Proteins c-raf - antagonists & inhibitors</topic><topic>Proto-Oncogene Proteins c-raf - metabolism</topic><topic>Pyrazoles - pharmacology</topic><topic>Quinones - pharmacology</topic><topic>Rifabutin - analogs & derivatives</topic><topic>Rifabutin - pharmacology</topic><topic>Technology, Pharmaceutical - methods</topic><topic>Technology, Pharmaceutical - trends</topic><topic>Transplantation, Heterologous</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hardcastle, Anthea</creatorcontrib><creatorcontrib>Boxall, Kathy</creatorcontrib><creatorcontrib>Richards, Juliet</creatorcontrib><creatorcontrib>Tomlin, Peter</creatorcontrib><creatorcontrib>Sharp, Swee</creatorcontrib><creatorcontrib>Clarke, Paul</creatorcontrib><creatorcontrib>Workman, Paul</creatorcontrib><creatorcontrib>Aherne, Wynne</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Assay and drug development technologies</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hardcastle, Anthea</au><au>Boxall, Kathy</au><au>Richards, Juliet</au><au>Tomlin, Peter</au><au>Sharp, Swee</au><au>Clarke, Paul</au><au>Workman, Paul</au><au>Aherne, Wynne</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90</atitle><jtitle>Assay and drug development technologies</jtitle><addtitle>Assay Drug Dev Technol</addtitle><date>2005-06</date><risdate>2005</risdate><volume>3</volume><issue>3</issue><spage>273</spage><epage>285</epage><pages>273-285</pages><issn>1540-658X</issn><eissn>1557-8127</eissn><abstract>High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.</abstract><cop>United States</cop><pmid>15971989</pmid><doi>10.1089/adt.2005.3.273</doi><tpages>13</tpages></addata></record> |
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subjects | Animals Benzoquinones Blotting, Western Carrier Proteins - antagonists & inhibitors Carrier Proteins - metabolism Cell Extracts - analysis Dose-Response Relationship, Drug Drug Design eIF-2 Kinase - antagonists & inhibitors eIF-2 Kinase - metabolism Enzyme Inhibitors - pharmacology HCT116 Cells Heterocyclic Compounds, 2-Ring - pharmacology HSP70 Heat-Shock Proteins - antagonists & inhibitors HSP70 Heat-Shock Proteins - metabolism HSP90 Heat-Shock Proteins - antagonists & inhibitors HSP90 Heat-Shock Proteins - metabolism HT29 Cells Humans Immunoassay - methods Lactams, Macrocyclic Lymphocytes - chemistry Lymphocytes - drug effects Lymphocytes - metabolism Mice Neoplasm Transplantation Proto-Oncogene Proteins c-raf - antagonists & inhibitors Proto-Oncogene Proteins c-raf - metabolism Pyrazoles - pharmacology Quinones - pharmacology Rifabutin - analogs & derivatives Rifabutin - pharmacology Technology, Pharmaceutical - methods Technology, Pharmaceutical - trends Transplantation, Heterologous |
title | Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90 |
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