title: New selective peptidyl di(chlorophenyl) phosphonate esters for visualizing and blocking neutrophil proteinase 3 in human diseases.
abstract: The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France,; Comprehensive Pneumology Center, Institute of Lung Biology and Disease, German Center for Lung Research (DZL), 81377 Munich and Max Planck Institute of Neurobiology, 82152 Planegg-Martinsried, Germany.
authors.forename: Carla
authors.initials: C
authors.lastname: Guarino
authors.affiliation: Faculty of Chemistry, University of Gdansk, 80-952, Gdansk, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Legowska
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Epinette
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS-Unité Mixte de Recherche (UMR),13288 Marseille, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Kellenberger
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Dallet-Choisy
authors.affiliation: Wroclaw University of Technology, Faculty of Chemistry, Division of Medicinal Chemistry and Microbiology, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Sieńczyk
authors.affiliation: Centre de Biophysique Moléculaire, UPR4301 CNRS, 45071 Orléans, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Gabant
authors.affiliation: Centre de Biophysique Moléculaire, UPR4301 CNRS, 45071 Orléans, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Cadene
authors.affiliation: Biotechnology Division, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece, and.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Zoidakis
authors.affiliation: Biotechnology Division, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece, and.
authors.forename: Antonia
authors.initials: A
authors.lastname: Vlahou
authors.affiliation: Faculty of Chemistry, University of Gdansk, 80-952, Gdansk, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wysocka
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Marchand-Adam
authors.affiliation: Comprehensive Pneumology Center, Institute of Lung Biology and Disease, German Center for Lung Research (DZL), 81377 Munich and Max Planck Institute of Neurobiology, 82152 Planegg-Martinsried, Germany.
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Jenne
authors.affiliation: Faculty of Chemistry, University of Gdansk, 80-952, Gdansk, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France.
authors.forename: Francis
authors.initials: F
authors.lastname: Gauthier
authors.affiliation: INSERM U-1100/EA-6305 Centre d'Etude des Pathologies Respiratoires and Université François Rabelais, 37032 Tours, France,. Electronic address: brice.korkmaz@inserm.fr.
authors.forename: Brice
authors.initials: B
authors.lastname: Korkmaz
mesh_terms: D000818:Animals; D017209:Apoptosis; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004952:Esters / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D006868:Hydrolysis; D007249:Inflammation; D007313:Insecta; D013058:Mass Spectrometry; D008956:Models, Chemical; D009154:Mutation; D053146:Myeloblastin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects; D009842:Oligopeptides / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D012997:Solvents
chemical_list: D004952:Esters; D009842:Oligopeptides; D063065:Organophosphonates; D010455:Peptides; D011480:Protease Inhibitors; D012997:Solvents; D011392:Proline; D053146:Myeloblastin
keywords: Activity-based Probes; Inflammation; Inflammatory Diseases; Neutrophil; Protease; Protease Inhibitor; Proteinase 3 (Myeloblastin); Serine Protease