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Improved fluorogenic histone deacetylase assay for high-throughput-screening applications
Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogenous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs co...
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| Published in: | Analytical biochemistry 2003-10, Vol.321 (2), p.202-208 |
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| Main Authors: | , , , |
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| Language: | English |
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| cites | cdi_FETCH-LOGICAL-c458t-12a965104772b3ab67304122ef432a9f193fd16e1e400bdaf1feedc468f78adf3 |
| container_end_page | 208 |
| container_issue | 2 |
| container_start_page | 202 |
| container_title | Analytical biochemistry |
| container_volume | 321 |
| creator | Wegener, Dennis Hildmann, Christian Riester, Daniel Schwienhorst, Andreas |
| description | Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogenous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs comprising an
ε-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery. |
| doi_str_mv | 10.1016/S0003-2697(03)00426-3 |
| format | article |
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ε-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery.</description><subject>Acetylation</subject><subject>Acetylpolyamine amidohydrolase</subject><subject>Amidohydrolases - pharmacology</subject><subject>Animals</subject><subject>Coumarins - chemistry</subject><subject>Drug Evaluation, Preclinical</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Fluorogenic substrates</subject><subject>Fluorometry - methods</subject><subject>High-throughput screening assay</subject><subject>Histone deacetylase</subject><subject>Histone Deacetylases - metabolism</subject><subject>Hydroxamic Acids - pharmacology</subject><subject>Kinetics</subject><subject>Liver - enzymology</subject><subject>Rats</subject><subject>Trypsin - metabolism</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><recordid>eNqFkE1v1DAQhq2qqF0KPwGUUwWH0Bk7dpITQis-KlXiQDlwsrz2eNcoGwc7qbT_Hre7gmNP72Ged0bzMPYG4QMCqpsfACBqrvr2HYj3AA1XtThjK4Re1SCgP2erf8gle5nzbwDERqoLdlkCUXVyxX7d7qcUH8hVflhiilsag612Ic9xpMqRsTQfBpOpMjmbQ-VjKtPtrp53KS7b3bTMdbaJSm3cVmaahmDNHOKYX7EX3gyZXp_yiv388vl-_a2--_71dv3prraN7OYauemVRGjalm-E2ahWQIOck29EGXnshXeoCKkB2Djj0RM526jOt51xXlyx6-Pe8sefhfKs9yFbGgYzUlyybmWLXCJ_FsSu40rKvoDyCNoUc07k9ZTC3qSDRtCP8vWTfP1oVpd8kq9F6b09HVg2e3L_WyfbBfh4BKj4eAiUdLaBRksuJLKzdjE8c-IvdjeVeA</recordid><startdate>20031015</startdate><enddate>20031015</enddate><creator>Wegener, Dennis</creator><creator>Hildmann, Christian</creator><creator>Riester, Daniel</creator><creator>Schwienhorst, Andreas</creator><general>Elsevier Inc</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>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20031015</creationdate><title>Improved fluorogenic histone deacetylase assay for high-throughput-screening applications</title><author>Wegener, Dennis ; Hildmann, Christian ; Riester, Daniel ; Schwienhorst, Andreas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c458t-12a965104772b3ab67304122ef432a9f193fd16e1e400bdaf1feedc468f78adf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Acetylation</topic><topic>Acetylpolyamine amidohydrolase</topic><topic>Amidohydrolases - pharmacology</topic><topic>Animals</topic><topic>Coumarins - chemistry</topic><topic>Drug Evaluation, Preclinical</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Fluorogenic substrates</topic><topic>Fluorometry - methods</topic><topic>High-throughput screening assay</topic><topic>Histone deacetylase</topic><topic>Histone Deacetylases - metabolism</topic><topic>Hydroxamic Acids - pharmacology</topic><topic>Kinetics</topic><topic>Liver - enzymology</topic><topic>Rats</topic><topic>Trypsin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wegener, Dennis</creatorcontrib><creatorcontrib>Hildmann, Christian</creatorcontrib><creatorcontrib>Riester, Daniel</creatorcontrib><creatorcontrib>Schwienhorst, Andreas</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>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wegener, Dennis</au><au>Hildmann, Christian</au><au>Riester, Daniel</au><au>Schwienhorst, Andreas</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improved fluorogenic histone deacetylase assay for high-throughput-screening applications</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2003-10-15</date><risdate>2003</risdate><volume>321</volume><issue>2</issue><spage>202</spage><epage>208</epage><pages>202-208</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><notes>ObjectType-Article-2</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-1</notes><notes>content type line 23</notes><notes>ObjectType-Article-1</notes><notes>ObjectType-Feature-2</notes><abstract>Histone deacetylases (HDACs) are key targets for chemotherapeutic intervention in malignant diseases. In this paper, a highly sensitive, nonisotopic, homogenous assay for high-throughput screening of HDAC inhibitors is presented. The assay is based on a new fluorogenic peptidic substrate of HDACs comprising an
ε-acetylated lysyl moiety and an adjacent 4-methylcoumarin-7-amide moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules are recognized as substrates by trypsin, which releases highly fluorescent 7-amino-4-methylcoumarin molecules in a subsequent step of the assay. The fluorescence increase is directly proportional to the amount of deacetylated substrate molecules, i.e., HDAC activity. Validation of an improved version of the assay revealed (i) a significantly lower enzyme consumption, (ii) an increased screening window coefficient, (iii) a good tolerance toward organic solvents, and (iv) a good suitability for a whole range of different HDAC-like enzymes. The novel assay thus will expedite studies of HDAC-like enzymes and in vitro screening for drug discovery.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>14511685</pmid><doi>10.1016/S0003-2697(03)00426-3</doi><tpages>7</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 2003-10, Vol.321 (2), p.202-208 |
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| source | ScienceDirect Freedom Collection |
| subjects | Acetylation Acetylpolyamine amidohydrolase Amidohydrolases - pharmacology Animals Coumarins - chemistry Drug Evaluation, Preclinical Enzyme Inhibitors - pharmacology Fluorescent Dyes - chemistry Fluorogenic substrates Fluorometry - methods High-throughput screening assay Histone deacetylase Histone Deacetylases - metabolism Hydroxamic Acids - pharmacology Kinetics Liver - enzymology Rats Trypsin - metabolism |
| title | Improved fluorogenic histone deacetylase assay for high-throughput-screening applications |
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