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Page 4 of 23 Skoreński et al. Rare Dis Orphan Drugs J 2023;2:6 https://dx.doi.org/10.20517/rdodj.2022.21

innate immune system. There are forms and releases in a process known as NETosis, a unique type of cell
death, after recognizing specific pathogens. NETs persistence is regulated by DNase I and DNase-like
proteins, which are responsible for maintaining a balance between the formation and degradation of
neutrophil extracellular traps [19,20] . Abnormal level of release NETs can be associated with the pathogenesis
of different disorders, including lung injury [19,21] , nephritis , cardiovascular disease , autoimmune
[23]
[22]
disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome [18,22] ), as well
as lung, breast or pancreatic cancer progression [24,25] . In the tumor microenvironment, the activity of release
NE from NETs may also correlate with the formation of breast or colorectal cancer metastases [26,27] .
Additionally, recent studies confirm that it is also related to COVID-19 due to SARS-CoV-2 infection can
directly induce the increased formation of NETs in neutrophils [28-30] .

The potent activity of NE is regulated by endogenous inhibitors, mainly by serpins such as α1-PI, α2-MG,
[15]
α1-antitrypsin (α1-AT), SLPI, and elafin . The uncontrolled outflow of HNE causes the degradation of
extracellular matrix components and destroys alveolar epithelial cells, which may lead to the development of
pulmonary disease . Accumulations and excessive HNE activation in the lung are associated with the
[17]
pathogenesis of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive
pulmonary disease, cystic fibrosis, bronchiectasis, or pneumococcal pneumonia. HNE is also involved in
rheumatoid arthritis due to the degradation of the matrix and destruction of cartilage components [12,15,17,31] .

HNE Synthetic substrates
The most common substrates used for routine assay of HNE activity, which are hydrolytically stable at
neutral pH, are chromogenic and fluorogenic peptide substrates. A synthesis of these simple compounds
was initially described in 1979. The values of K , k , and k /K for the most potent chromogenic substrate
cat
M
cat
M
MeO-Suc-Ala-Ala-Pro-Val-pNA (λ = 320 nm, λ = 490 nm) are respectively 0.14 mM, 17 s ,
-1
ex
em
12 × 10 M s , and for fluorogenic substrate MeO-Suc-Ala-Ala-Pro-Val-AMC (λ = 370 nm, λ = 460 nm)
-1 -1
4
ex
em
-1 -1[32]
the same values are 0.29 mM, 3.3 s and 11 × 10 M s .
3
-1
Wysocka et al. have described the synthesis and characterization of several fluorescent substrates displaying
[33]
FRET for HNE . The best parameters were obtained for PEG-FAM-Ala-Pro-Glu-Glu-Ile-Met--Asp-Arg-
Gln-BAD [where PEG = 2-(2-(2-aminoethoxy)ethoxy)acetic acid, FAM = Lys(Fam)-OH, Fam = 5(6)-
-1
fluorescein, BAD = Ala(Bad)-OH], with the K of 24.2 ± 3.1 µM, k of 6.2 ± 0.9 s , and k /K of
M
M
cat
cat
-1 -1[33]
37.9 × 10 M s .
4
In 2013 Sun et al. described a design and simple synthesis of a pentafluoroethyl conjugated 7-amino-4-
[34]
trifluomethylcoumarin (AFC) as the first non-peptide-based fluorescent probe and a substrate for NE .
This low molecular weight compound, 2,2,3,3,3-pentafluoro-N-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-
yl) propenamide, with high specificity (K = 20.45 ± 1.85 µM, k = 21.84 ± 4.37 min ,
-1
M
cat
k /K = 1.07 μM min ) was a promising candidate for tracing elastase’s activity in human serum. However,
-1
-1
cat
M
the green fluorescence of this fluorophore limited its use in tests on cells and animal models .
[34]
HNE ABPs and Inhibitors
Prolastin®, a purified α1-antitrypsin, is the first neutrophil elastase inhibitor available on the market used in
the treatment of α1-AT deficiency . Elaspol®, also known as sivelestat or ONO-5046 (1), is the second NE
[15]
inhibitor approved for use and the first non-peptide inhibitor. Silvelestat is applied in Japan and South
Korea for the treatment of ALI and ARDS associated with systemic inflammatory response syndrome. This
molecule is known as a highly specific and effective competitive NE inhibitor with IC and K of 44 nM and
50
i
[35]
200 nM . Alvelestat, AZD9668 (2), is a reversible oral highly selective NE inhibitor with IC and K of
i
50
12 nM and 9.4 nM. This inhibitor is currently in phase II of clinical trials to treat patients with bronchiolitis