A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate.
The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, 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 neutrophil elastase 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 (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.
INSERM U-1100 Pathologies Respiratoires, Protéolyse et Aérosolthérapie, Faculté de Médecine, Université François Rabelais, 37032 Tours, France.
Christophe
C
Epinette
Cécile
C
Croix
Lucie
L
Jaquillard
Sylvain
S
Marchand-Adam
Christine
C
Kellenberger
Gilles
G
Lalmanach
Martine
M
Cadene
Marie-Claude
MC
Viaud-Massuard
Francis
F
Gauthier
Brice
B
Korkmaz
D000595:Amino Acid Sequence; D056649:Cathepsin G / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015195:Drug Design; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D053146:Myeloblastin / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011014:Pneumonia / Q000188:drug therapy / Q000201:enzymology*; D011485:Protein Binding; D053491:Proteinase Inhibitory Proteins, Secretory / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D059748:Proteolysis; D013183:Sputum / Q000201:enzymology; D013379:Substrate Specificity; D013997:Time Factors
D009842:Oligopeptides; D010455:Peptides; D053491:Proteinase Inhibitory Proteins, Secretory; C571296:azapro-3 peptide; D056649:Cathepsin G; D019272:Leukocyte Elastase; D053146:Myeloblastin