title: In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics.
abstract: 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.
authors: Center for Proteomic Chemistry/Expertise Platform Proteases, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.
authors: Ludovic C J
authors: LC
authors: Gillet
authors: Kenji
authors: K
authors: Namoto
authors: Alexandra
authors: A
authors: Ruchti
authors: Sjouke
authors: S
authors: Hoving
authors: Danielle
authors: D
authors: Boesch
authors: Bruno
authors: B
authors: Inverardi
authors: Dieter
authors: D
authors: Mueller
authors: Michele
authors: M
authors: Coulot
authors: Patrick
authors: P
authors: Schindler
authors: Patrick
authors: P
authors: Schweigler
authors: Anna
authors: A
authors: Bernardi
authors: Shirley
authors: S
authors: Gil-Parrado
mesh_terms: D018938:Caco-2 Cells; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D008954:Models, Biological; D063065:Organophosphonates / Q000494:pharmacology; D020543:Proteome / Q000032:analysis / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D063065:Organophosphonates; D020543:Proteome; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors