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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M700505-MCP200v1 7/7/1241    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gillet, L. C. J. Articles by Gil-Parrado, S. Search for Related Content PubMed PubMed Citation Articles by Gillet, L. C. J. Articles by Gil-Parrado, S. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 7:1241-1253, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

In-cell Selectivity Profiling of Serine Protease Inhibitors by Activity-based Proteomics^*,S

Ludovic C. J. Gillet, Kenji Namoto, Alexandra Ruchti, Sjouke Hoving, Danielle Boesch, Bruno Inverardi, Dieter Mueller, Michele Coulot^¶, Patrick Schindler^¶, Patrick Schweigler, Anna Bernardi and Shirley Gil-Parrado^,||

From the Center for Proteomic Chemistry/Expertise Platform Proteases, Developmental and Molecular Pathways, and ^¶ Biologics Center, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.

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