title: Clickable 4-Oxo-β-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes.
abstract: Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC, or 'click' chemistry) approach based on the 4-oxo-β-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure-activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-β-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC50 values in the nanomolar range (56-118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools.
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors.forename: Eduardo F P
authors.initials: EF
authors.lastname: Ruivo
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors.forename: Lídia M
authors.initials: LM
authors.lastname: Gonçalves
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors.forename: Luís A R
authors.initials: LA
authors.lastname: Carvalho
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors.forename: Rita C
authors.initials: RC
authors.lastname: Guedes
authors.affiliation: Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157, Oeiras, Portugal.|Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, 1030, Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hofbauer
authors.affiliation: Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157, Oeiras, Portugal.
authors.forename: José A
authors.initials: JA
authors.lastname: Brito
authors.affiliation: Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157, Oeiras, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Archer
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. rmoreira@ff.ulisboa.pt.
authors.forename: Rui
authors.initials: R
authors.lastname: Moreira
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. sdlucas@ff.ulisboa.pt.
authors.forename: Susana D
authors.identifier: 0000-0002-5555-5594
authors.initials: SD
authors.lastname: Lucas
mesh_terms: D057930:Click Chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015394:Molecular Structure; D053491:Proteinase Inhibitory Proteins, Secretory / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D020543:Proteome / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D047090:beta-Lactams / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D053491:Proteinase Inhibitory Proteins, Secretory; D020543:Proteome; D047090:beta-Lactams; D019272:Leukocyte Elastase
keywords: 4-oxo-β-lactams; activity-based probes; click chemistry; fluorescent probes; human neutrophil elastase