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Skoreński et al. Rare Dis Orphan Drugs J 2023;2:6 https://dx.doi.org/10.20517/rdodj.2022.21 Page 15 of 23

-3
-1 -1
Pro-Lys-pNA (33) (K = 2.75 × 10 M and k /K 1,483 M s ).
M
M
cat
In 2007, Wysocka et al. presented studies about new chromogenic substrates of CatG . The combinatorial
[75]
chemistry methods enable the production of new, sensitive cathepsin G substrates. The introduction of the
non-proteinogenic amino acid residue (4-guanidine-L-phenylalanine) in position P1 increases activity twice
as high as in the case of Phe (Ac-Phe-Val-Thr-Gnf-Anb-NH (34) K = 203 µM and k /K 95,300 M s ;
-1 -1
M
cat
2
M
-1 -1
Ac-Phe-Val-Thr-Phe-Anb-NH K = 464 µM and k /K 7,900 M s ). The additionally obtained substrate is
M
2
M
cat
not cleaved by proteinase 3, human leukocyte elastase or chymotrypsin. A further modification by replacing
the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence
donor and substrate elongation led to the Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH (35), the sequence with
2
3
a specificity constant (K = 2 µM, k /K = 252 × 10 M s ) which is two orders of magnitude higher than
-1 -1
M
M
cat
[76]
that of the parent compound. The detection limit of CatG using this substrate is 70 pM . Later in 2019,
Groborz et al. developed new fluorogenic substrates of CatG based on a fluorescence quenching
[77]
-1 -1
mechanism . The most active inhibitor from their studies was OS-CG_11 (36) with k /K = 206,854 M s
M
cat
[Figure 17]. This substrate was also active against PR3 and displayed k /K 146,824 M s .
-1 -1[77]
M
cat
CatG ABPs and Inhibitors
In 1991, two phosphonic inhibitors of CatG were reported . The analog of phenylalanine: Cbz-Phe (OPh)
[78]
P
2
-1 -1
P
(37) and phenylglycine: Cbz-Phg (OPh) (38) displays similar poor activity against CatG (k /I = 76 M s
obs
2
and k /I = 91 M s respectively) [Figure 18]. Further incorporation of the basic functional group into the
-1 -1
obs
aromatic side chain together with phenyl esters modification and peptide chain elongation resulted in the
inhibitor Ac-Phe-Val-Thr-(4-guanidine)Phg (OC H -4-S-Me) (39) with k /I = 256,000 M s
-1 -1[79]
P
obs
2
6
4
[Figure 18]. Based on this inhibitor, Grzywa et al. developed a low-molecular-weight activity-based probe:
P
-1 -1
Bt-LC-Suc-Phe-Val-Thr-(4Gu)Phg (O-C H -4-S-CH ) (40) [Table 2] with k /I = 240 M s which
3 2
4
6
obs
displayed absolute specificity toward CatG in western blotting analysis among two other neutrophil
proteases- HNE and PR3 [Figure 18]. Another phosphonate base ABPs was reported by the Drag group in
[64]
2017 . From a series of compounds with a different fluorophore inhibitor 202 (41), it showed the best
[65]
potency with 43,000 M s [Figure 18]. The inhibitor 202 displayed high selectivity and did not react with
-1 -1
other neutrophil serine proteases.
In 2015, Serim et al. reported quenched fluorescent activity-based probes based on mixed alkyl-aryl
phosphonate esters [Figure 18]. Compound 15 (42) could label the CatG in SDS-PAGE gel.
[80]
Isocoumarins equipped with detection tags were reported as CatG ABPs. Compound BIC5 showed
-1 -1
k /I = 59 M s against CatG. BIC5 (43) was not specific towards CatG and could inhibit chymotrypsin and
obs
HNE (k /I = 260 M s and 96,000 M s respectively) [Figure 18]. In 2012, the Verhelst research group
-1 -1
-1 -1
[81]
obs
reported clickable isocumarin-based compounds that act as ABPs of serine proteases . Compound IC14
[82]
(44) could label active CatG in lysates of mammalian cells (EL4 mouse lymphoma). The detection limit was
0.03% of total protein [Figure 18].
Recently, Kahler et al. developed aminomethyl phosphinate esters as inhibitors of serine protease. This
group of compounds can interact with prime enzyme site. Primary compound testing revealed that
[83]
inhibitor 13a (45) can react with CatG [Figure 18].
NSP4
Human NSP4 (encoded by the gene PRSS57) is a trypsin-fold protease stored in neutrophil azurophilic
granules [84,85] . Since the NSP4 discovery in 2012 by the D.E.Jenne research group, numerous studies