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

Neutrophils are activated by the emergence of specific danger signals such as inflammatory cytokines,
molecules derived from pathogens, or a host tissue damage signal, and rapidly relocate from the peripheral
blood into the inflammatory or damaged sites. As a part of the immune system’s first line of defense,
neutrophils employ various mechanisms in order to eliminate the invading pathogens as well as to regulate
inflammatory processes, including chemotaxis, phagocytosis, the release of reactive oxygen species (ROS)
generated by the NADPH oxidase, formation of extracellular chromatin filaments containing granule-
derived proteins (named neutrophil extracellular traps; NETs), and degranulation . However, these
[1,2]
protective mechanisms can also be destructive to host cells; therefore, neutrophil production, maturation,
distribution, and disposal need to be strictly regulated .
[3]

Neutrophil granules contain numerous proteolytic enzymes of four different classes of proteases: cysteine
proteases (e.g., cathepsin C), aspartyl proteases (e.g., cathepsin D, cathepsin E), metalloproteases
(e.g., collagenases, gelatinases), and serine proteases. So far, four active neutrophil serine proteases (NSPs)
have been identified: neutrophil elastase (NE), cathepsin G (CatG), proteinase 3 (PR3), the recently
discovered neutrophil serine protease 4 (NSP4), and azurocidin (CAP-37), an inactive serine protease
displaying an antimicrobial activity . The active NSPs also play an important role in the antimicrobial
[4]
activity of neutrophils. They are involved in the degradation of pathogens and the regulation of
inflammatory responses. NSPs are chymotrypsin-like serine proteases that contain a conserved serine
residue in the catalytic triad His -Asp -Ser (chymotrypsin numbering), and the hydroxyl group of this
195
57
102
residue is responsible for performing a nucleophilic attack on the carbonyl carbon of the scissile peptide
bond [Figure 1]. The proteolytic activity of release NSPs at the inflammatory sites is regulated by the
[5]
presence of several endogenous inhibitors, members of the serpin family, including α2-macroglobulin (α2-
MG), α1-protease inhibitor (α1-PI), secretory leucoprotease inhibitor (SLPI), leucocyte elastase inhibitor
(LEI), and elafin [4,6-8] .

Neutrophil serine proteases are synthesized as inactive zymogens in the bone marrow during early
granulocyte development and contain a dipeptide structure at the N-terminal. These zymogens undergo
maturation, which activates the enzymes and produces active neutrophil serine proteases. This N-terminal
proteolytic processing of the inactive pre-form of NSPs takes place in the endoplasmic reticulum and
depends on the activity of dipeptidyl peptidase I (DPPI). Dipeptidyl peptidase I, aminodipeptidase, also
[5,9]
known as cathepsin C, is a cysteine protease located in the primary granules . Under normal physiological
conditions, the activity of NSPs must be controlled in order to avoid the development of pathological states
or autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, chronic respiratory
diseases (e.g., chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis), and cancer [10-14] .

HUMAN NEUTROPHIL ELASTASE
Human neutrophil elastase (HNE), also known as leukocyte elastase (EC 3.4.21.37), is a globular
glycoprotein belonging to the chymotrypsin family. HNE is located in neutrophils, eosinophils, mast cells,
monocytes, keratinocytes, and fibroblasts. This 29 kDa protease consists of a single polypeptide chain of 218
amino acids and two asparagine-linked carbohydrate chains localized at Asn95 and Asn144. The presence of
four disulfide bridges and 19 arginine residues helps stabilize the structure, resulting in basic properties and
an isoelectric point around 10-11 [Figure 2]. The primary structure of HNE exhibits homology with other
NSPs, PR3 (57%) and CatG (37%). The substrate specificity of HNE is very similar to PR3, with both
enzymes cleaving substrates after small aliphatic residues (Ala, Val, GABA, and norVal) .
[15]

This enzyme has a broad physiological function, including the degradation of elastin and other extracellular
proteins like collagen (type I-IV), fibronectin, laminin and proteoglycans, and it has coagulation factors