Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

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dc.contributor.authorDeu, Edgaren
dc.contributor.authorYang, Zhimouen
dc.contributor.authorWang, Floraen
dc.contributor.authorKlemba, Michaelen
dc.contributor.authorBogyo, Matthewen
dc.contributor.departmentBiochemistryen
dc.date.accessioned2018-11-12T20:11:11Zen
dc.date.available2018-11-12T20:11:11Zen
dc.date.issued2010-08-05en
dc.description.abstractBackground High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1371/journal.pone.0011985en
dc.identifier.eissn1932-6203en
dc.identifier.issue8en
dc.identifier.othere11985en
dc.identifier.pmid20700487en
dc.identifier.urihttp://hdl.handle.net/10919/85819en
dc.identifier.volume5en
dc.language.isoenen
dc.publisherPLOSen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleUse of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extractsen
dc.title.serialPLOS ONEen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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