An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide

Natacha Jugniot (1) , Indranil Duttagupta (2) , Angelique Rivot (1) , Philippe Massot (1) , Colleen Cardiet (1) , Anne Pizzoccaro (3) , Marion Jean (4) , Nicolas Vanthuyne (4) , Jean-Michel Franconi (1) , Pierre Voisin (1) , Gilles Devouassoux (3) , Elodie Parzy (1) , Eric Thiaudiere (1) , Sylvain R.A. Marque (2, 5) , Abderrazzak Bentaher (3) , Gérard Audran (2) , Philippe Mellet (1)

1 CRMSB - Centre de résonance magnétique des systèmes biologiques
2 ICR - Institut de Chimie Radicalaire
3 PI3 - Physiopathologie de l'immunodépression associée aux réponses inflammatoires systémiques - EA 7426
4 ISM2 - Institut des Sciences Moléculaires de Marseille
5 RAS, SB, NN - Vorozhtsov Novosibirsk Institute of Organic Chemistry

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 K-m = 15 +/- 2.9 mu M, k(cat)/K-m = 930,000 s(-1) M-1 and K-m = 25 +/- 5.4 mu M, k(cat)/K-m = 640,000 s(-1) M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concen trations 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.

Domains: Chemical Sciences

Natacha Jugniot, Indranil Duttagupta, Angelique Rivot, Philippe Massot, Colleen Cardiet, et al.. An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide. Free Radical Biology and Medicine, 2018, 126, pp.101--112. ⟨10.1016/j.freeradbiomed.2018.08.006⟩. ⟨hal-02091886⟩