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A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate
Author(s) -
Korkmaz Brice,
Kellenberger Christine,
Cadène Martine,
Viaud-Massuard Marie-Claude,
Gauthier Francis
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.851.10
Subject(s) - proteinase 3 , cathepsin g , neutrophil elastase , elastase , proteases , chemistry , proteolysis , enzyme , substrate (aquarium) , biochemistry , azurophilic granule , neutrophile , inflammation , substrate specificity , biology , immunology , myeloperoxidase , in vitro , ecology
The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural and functional resemblance to neutrophil elastase (NE). Thus, all natural inhibitors of PR3 preferentially target NE. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly NE or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl‐enzyme complex; it is a reversible competitive inhibitor with a K i comparable to the K m of the parent substrate. Low concentrations (10 μM) of the azapeptide (azapro‐3) totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in crude biological samples. Azapro‐3 resisted proteolysis by its target enzyme, by the proteases in neutrophils homogenates and by those in lung secretions of patients with neutrophilic lung inflammation for hours.