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.
Degradome analysis indicates that protease and protease inhibitor genes represent more than 2% of total genes in human genome [
NE (EC 3.4.21.37) is a 29 kDa serine protease of chymotrypsin family stored in azurophilic granules of polymorphonuclear neutrophils [
A standard approach for looking at proteases in inflamed situations is the analysis of their transcript levels. Because of the posttranslational modifications, monitoring protease activity directly is more reliable to translate its roles in biological events. Another commonly used method for the quantification of NE is the immunodiagnostic from biological fluid samples. Antibody-related techniques yield information on total protease amount but lack the ability to differentiate between active and inactive enzyme forms. Methods to overcome such limitations tried to combine classical ELISA with the application of active-site inhibitors. A colorimetric active site-specific immunoassay (CASSIA) was described [
Clinical symptoms are often undetectable at early stage of a disease, making diagnosis approach even more challenging. Nowadays, no reliable clinical methods exist to image deleterious NE proteolytic activity, which is again convincingly documented as a culprit in tissue destructive diseases. To address this concern, the field of functional molecular imaging represents an attractive and relevant tool [
This review will discuss the design of novel probes and how their application in NE proteolytic activity imaging could become a reliable clinical diagnostic tool. On one hand, imaging agents binding their biological target with high specificity and affinity are worked out, including the design of substrate-based probes and activity-based probes. On the other hand, imaging instruments including optical imaging and Magnetic Resonance Imaging (MRI) methods should be able to detect disorders with high sensitivity and high resolution (Figure
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.
The development of functional imaging technologies has led to the production of a myriad of molecular imaging agents for a variety of proteases [
Recapitulative set of probes for NE proteolytic activity imaging.
Probe sequence |
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Nature of probe | Detection modality | Probe name | References |
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99mTC-NX21909 | 2 × 105 ( |
Activity-based probe | Positron emission tomography | NX21909 | [ | ||
99mTC-MAG3-EPI-HNE-2 | 2 × 10−6 ( |
Activity-based probe | Positron emission tomography | EPI-HNE-2 | [ | ||
Biotin-PEG(4)-Nle( |
1.4 × 107 ( |
n.d | n.d | Activity-based probe | Optic (fluorogenic) | Elastase-PK101 | [ |
Ac-AAPV-AMC | 5.8 × 103 | n.d | n.d | Substrate-based probe | Optic (fluorogenic) | — | [ |
MeO-Suc-AAPV-AMC | 11 × 103 | 290 | 3.3 | Substrate-based probe | Optic (fluorogenic) | — | [ |
CFP-TSGGSGGTRQFIRWGGGGSGGTTG-YFP-HHHHHH | 390 × 105 ( |
0.7 ± 0.2 | 27 ± 5.4 ( |
Substrate-based probe | Optic (fluorogenic) | Protein Biosensor IV | [ |
Abz-QPMAVVQSVPQ-EDDnp | 10.9 × 105 | n.d | n.d | Substrate-based probe | Optic (fluorogenic) | NEmo-1 & NEmo-2 | [ |
Neutrophil Elastase 680 FAST™ | [ | ||||||
CNC-(O-C(O)G-NHC(O)-Suc-APA-AMC | 33.5 × 105 | n.d | n.d | Substrate-based probe | Optic (fluorogenic) | PepNA | [ |
MeO-Suc-AAPV-(R/S)C12H23NO5P▪ | Substrate-based probe | MRI (dynamic nuclear polarization) | — | [ | |||
|
9.3 × 105 | 15 ± 2.9 | 14 ± 0.9 | ||||
|
6.4 × 105 | 25 ± 5.4 | 16 ± 1.1 |
n.d., no data.
Activity-based probes (ABPs) are low-molecular-weight molecule reporters designed to covalently bind a target enzyme as an active site reacting inhibitor and allow to visualize and localize active protease using fluorescence-based imaging modalities. All ABPs share a similar basic design, which incorporates elements required for targeting, modification, and detection of target proteins (Figure
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
ABPs appear very interesting for living organisms imaging applications. Indeed, one of the advantages of ABPs relies in the fact that the selectivity can be controlled both by the warhead and linker sequence. In this aim, focus on warhead and linker is more suitable over reporter tag.
Numerous peptide-based as well as nonpeptidyl inhibitors have been studied for NE specific studies [
In addition to warhead development, recognition sequence of the linker adds to the probe specificity. This link controls the specificity of the inhibitor toward its target by a recognition element such as a peptidic sequence. The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, obviously limiting the degree of conformational space that can be surveyed. In their studies, Kasperkiewicz and coworkers reported the design of a hybrid natural and nonnatural peptidic substrate of NE, PK101, using a hybrid combinatorial substrate library profiling [
Although the development of selective ABP probes remains a challenge, we believe that incorporation of novel warheads mixed with original substrate design will enable a very specific targeting of biomarkers as NE. Nevertheless, by nature, ABPs bind only a single protease and generate only one detectable molecule per binding event. The detected signal is then directly proportional to the overall concentration of active protease, and detection of low-abundance proteins may be challenging.
Signal amplification by multiple processing events can be successfully achieved by substrate-based probes. Indeed, one of the major benefits of using the turnover of substrate as a reporter is that a single active protease can process many substrates continuously leading thereby to signal amplification [
Molecular imaging requires high-resolution and highly sensitive instruments to detect imaging agents that connect the imaging signal with the molecular event. Molecular imaging is easily performed using fluorescent probes enabling 3D images on small animals. A variety of organic fluorophores with emission wavelengths ranging from visible to near infrared region have been synthetized. These molecules can be modified with additional groups to optimize their inherent properties such as photophysical characteristics, solubility, cell permeability, toxicity, or enzyme specificity. Currently, three major types of activated fluorescent probes are used to monitor NE activity (Figure
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.
Potential utilization of FRET-based probes to monitor protease activity has been investigated in 2004 by Felber and colleagues [
Other strategy consists in quenched FRET probes with improved specificity toward human and mouse neutrophil elastase, with the substrate sequence PMAVVQSVP [
So far, studies of NE activity have focused on free NE form. Interestingly, several reports demonstrated [
A widely used series of probes is based on poly-
In 2005, Edwards and coworkers outlined an approach involving the use of cotton cellulose nanocrystal (CNC) fluorescent peptide conjugates as a support for a sensitive biosensor for NE and porcine pancreatic elastase (CNC-(O-C(O)Gly-NHC(O))succinyl-Ala-Pro-Ala-AMC) [
There are however several drawbacks of using optical imaging: (a) substrate fluorescence quenching is not complete hence requiring long waiting times to eliminate nonspecific “blinding” light, (b) light tissue penetration is limited and prevents imaging of deeply seated tissues or skull, and (c) three-dimensional images are obtained by reconstruction.
MRI appears particularly well suited to deliver exquisite anatomical details. It has a superior true 3D coding along with exceptionally good soft tissue contrast compared to optical imaging. MRI offers high spatial resolution and an unlimited depth penetration. Completely noninvasive, it allows simultaneous acquisition of anatomical structure and physiological function, particularly relevant for longitudinal follow-up involving multiple acquisitions. Nevertheless, the use of MRI is hampered by the limited sensitivity so far prevented clinical molecular imaging such as enzyme activity imaging. Thus, it requires the design of smart contrast agents and development of powerful signal amplification strategies.
After such proof-of-concept, a
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.
The rapid expansion of molecular imaging technologies highlights promising prospects for early diagnosis of proteolysis. Substrate-based imaging agents have been recently shown to have strong values for NE imaging as biomarker of inflammation. Significantly, strategies using substrate coupled to an original nitroxide in OMRI exhibit multiple advantages. Totally noninvasive, OMRI values are highly resolved and highly sensitive. Recently, another type of nitroxide was used as theranostic approach for the treatment of solid tumors. This smart agent named “Alkoxynamine” can, in vitro, spontaneously undergo hemolysis producing a highly reactive alkyl agent which in turn would induce cancer cell death and a stable nitroxide which would serve as imaging contrast agent by OMRI [
Application to human diagnosis will require further development in terms of specificity and localization. Unnatural amino acid-recognition sequences could overcome such concerns by enhancing the specificity of proteolysis. On the other hand, ABPs, as covalently linked to the target protease (NE), lead to a prolonged retention at the inflamed site. This property contrasts with the irremediable diffusion of substrate-based probe.
Hence, the use of NE probes may ultimately lead to an easy methodology consisting in new diagnostic tools functioning through noninvasive protocols [
In addition to clinical diagnosis, evaluation of disease severity and follow-up, NE molecular imaging would also certainly have a huge potential in drug development improvement [
Although overall the molecular imaging is still at stage of development, it is expected that more advancements will be achieved in the area of molecular imaging agent, and in near future, molecular imaging techniques should drive clinical transformations. By building a complete inventory of endangered areas, proteolysis imaging would allow to respond actively in a way that leads to the optimum outcome for the patient when organs can still be preserved [
Activity-based probe
7-Amino-4-methylcoumarin
Bronchoalveolar lavage
Chemical exchange saturation transfer
Cystic fibrosis
Cyan fluorescent protein
Chloromethyl ketone
Cellulose nanocrystal
Chronic obstructive pulmonary disorder
Enzyme commission
Electronic paramagnetic resonance
Förster resonance energy transfer
Magnetic resonance imaging
Neutrophil elastase
Neutrophil extracellular trap
Overhauser-enhanced magnetic resonance imaging
Peptide-nanocellulose aerogel
Proteinase 3
Serine 195
Yellow fluorescent protein.
The authors declare that they have no conflicts of interest.
This work was supported by the French ANR PULMOZYMAGE (ANR-15-CE18-0012-01).