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Page 18 of 23 Skoreński et al. Rare Dis Orphan Drugs J 2023;2:6 https://dx.doi.org/10.20517/rdodj.2022.21

which is stabilized by a solvent-exposed H-bond network. Interestingly, except for NSP4, all other trypsin-
like proteases rely heavily on the salt bridge within a/the well-defined S1 pocket. As a result of the unusual
substrate recognition mechanism, NSP4 can hydrolase substrates with methylarginine or citrulline at the P1
position. This suggests one of the possible roles played by NSP4 protease, which is processing the post-
translationally modified proteins. The substrate used during the studies was MCA-PEG-Ile-Arg-Arg-Ser-
-1 -1
Ser-Tyr-Ser-Phe-Lys (Dnp)-Lys (K = 13.8 µM and k /K 10,000 M s ).
M
cat
M
The first studies to deeply investigate the substrate preferences of NSP4 were made by Kasperkiewicz
et al. . They synthesized the library of fluorogenic substrates with a fixed arginine residue at the P1
[89]
position. Activity studies showed that at the P2 position, substrates with proline residue were the most
active among proteinogenics aminoacids. The substrates with the proline analog -Oic were the most active.
P3 position revealed broad tolerance where Val, Ile, Tyr, Arg, Lys and Phe were the most active. The best
non- proteinogenic amino acid was the phenylalanine derivative with a guanidine group in a/the para
position (Phe(guan)). Homocyclohexylalanine (hCha) was found as the optimal amino acid at the P4
position. The most active substrate from the studies was Ac-hCha-Phe(guan)-Oic-Arg-ACC
(k /K = 32,000 M s ) (PK421) (46) [Figure 19]. Substrate PK421 is only very weakly hydrolyzed by CatG
-1 -1
M
cat
(k /K = 477 M s ).
-1 -1
cat
M
[90]
One year later, Wysocka et al. presented a new library of the PEGylated substrates of NSP4 protease .
From the library of a novel type of peptidomimetics composed of diaminopropionic acid residues modified
with structurally diverse heterobifunctional polyethylene glycol chains (DAPEG), substrate 3 (47) was the
most active with (K = 7.6 µM and k /K 13,1579 M s ) [Figure 19].
-1 -1
M
cat
m
Inhibitors and ABPs
Jenne et al. found that among natural inhibitors of NSPs - NSP4 was most efficiently inhibited by
antithrombin-heparin. α1-proteinase inhibitor (α1-antitrypsin) and C1 inhibitor can block enzymatic
[84]
activity of NSP4 .
The first synthetic inhibitor and ABP were reported by Kasperkiewicz et al. . The substrate conversion into
[89]
the diphenyl phosphonate probe with a biotin tag led to the Biot-Ahx-hCha-Phe(guan)-Oic-ArgP(OPh)
2
(48) (k /I = 3.8 × 10 M s against NSP4 and k /I = 3.0 × 10 M s against CatG and inactive against PR3
-1 -1
6
3
-1 -1
obs
obs
[89]
an HNE) [Figure 19].
Further studies by Kasperkiewicz et al. about the development of the fluorescent ABPs of NSP lead to the
[65]
compounds which are related to 401 and equipped with different N-Terminal fluorescent tags . ABP with
-1 -1
3
6
BODIPYFL fluorophore (49) was the most active with k /I = 3.8 × 10 M s , k /I = 3.0 × 10 M s
-1 -1
obs
obs
[Figure 19].
SUMMARY
NSPs are protein-degrading enzymes but also take part in a wide variety of pathophysiological processes.
Thus, their inhibitors are considered potential therapeutics. Despite the fact of extensive research on
substrates, inhibitors and ABPs of NSPs, there is no FDA-approved drug acting as an NSPs inhibitor.
The most advanced research was done in the field of HNE inhibitors, where inhibitors of this enzyme
entered clinical trials. However, clinical trials of HNE inhibitors have faced challenges and failures. For
example, a phase II clinical trial of an HNE inhibitor called AZD9668 (Alvelestat) in patients with COPD
failed to show significant improvement in lung function or other clinical endpoints compared to a placebo.