Neutrophil Elastase Activity Imaging: Recent Approaches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes

Abstract

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

Figure 1

Key elements necessary for neutrophil elastase (NE) proteolytic imaging. The concept is to integrate molecular biomarker, chemical probes with imaging instruments to visualize, localize, and quantify NE activity for diagnosis (disease initiation and/or progression) and therapy follow-up of inflammatory processes. OPTIC: optical imaging; MRI: magnetic resonance imaging.

Figure 2

Overall principle of activity-based probe (ABP). Warhead (grey triangle) structurally matches with the target protease (purple). Active ABP can be detected by the tag (blue star). AA₁-AA_n indicates amino acid position in the specific peptide.

Figure 3

Protease-sensitive probes for optical imaging. (a) Fluorogenic (F)/chromogenic (C) enzyme-sensitive probe. One fluorescent or chromogenic molecule is bound to a peptide. Spectroscopic properties will be altered upon proteolysis. (b) Fluorophore-quencher type probe. Förster resonance energy transfer- (FRET-) based probes require a donor and an acceptor fluorophore pair each saturated on one side of the enzyme cleavage site. (c) Polymeric-peptide conjugate probe. Overabundance of fluorophores coupled to a polymer backbone via a peptide substrate. Black arrowheads depict cleavage site within the amino acid sequence.

Figure 4

Proteolysis imaging by MRI. Proteolysis of peptide-locked nitroxide (1) into a free nitroxide (2) by neutrophil elastase creating high contrast in vivo by OMRI and EPR shift in vitro. Black arrowhead designates cleavage site in the amino acid specific sequence.