LAUSR

Abstract Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non‐invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO‐Suc‐(Ala)2‐Pro‐Val‐nitroxide, a line‐shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser‐enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ± 2.9 &mgr;M, kcat/Km = 930,000 s−1 M−1 and Km = 25 ± 5.4 &mgr;M, kcat/Km = 640,000 s−1 M−1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho‐alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear‐cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non‐infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection. Graphical abstract Figure. No caption available. HighlightsWe synthetized a peptide‐linked radical specific to elastase and proteinase 3.A significant shift in hyperfine coupling constants is obtained upon proteolysis.Unpaired electron is used to detect enzyme activity by Overhauser‐Enhanced MRI.Imaging elastase activity is a step to inflammation diagnosis.