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Stojkovska Docevska et al. Rare Dis Orphan Drugs J 2023;2:14 https://dx.doi.org/10.20517/rdodj.2023.09 Page 7 of 17

[90]
polymerization of LLOMe accumulated in lysosomes into its membranolytic polymeric form .

In vivo, cathepsin C activity is regulated by endogenous proteinaceous inhibitors. Like other PLP, it is
amendable to inhibition by inhibitors from the cystatin family [91,92] . Of note, it is also inhibited by cystatin
[94]
[93]
F , which is expressed only in immune cells . Interestingly, inhibition depends on the proteolytic
processing of cystatin F to remove its N-terminal part, which cannot be accommodated into the active site
[93]
of cathepsin C . Recently, it was found that Glu-Lys and Gly-Glu dipeptides derived from the pro-region
of granzymes A and B, respectively, inhibit the transferase activity of cathepsin C with IC values < 20 mM
50
at pH 7.4 and peptidase activity at pH 5.5 with K values of 20 mM and 2.5 mM for Glu-Lys and Gly-Glu,
i
respectively . Although this finding is unlikely to be considered as a strategy for pharmacological targeting
[95]
of cathepsin C, it is one of the few documented examples of product inhibition in this family of peptidases
and provides additional insight into the diversity of cathepsin C regulation in vivo.

PHARMACOLOGICAL INHIBITION OF CATHEPSIN C
Because of its critical roles in the activation of neutrophil and other serine peptidases, cathepsin C is
considered a promising target for the treatment of diseases associated with overactivity of cells producing
and secreting these peptidases, such as chronic inflammatory diseases, autoimmune diseases, and so on.
Cathepsin C is by no means an easy target in these diseases, as successful anti-cathepsin C therapies must
achieve a sustained inhibitory effect directly in the bone marrow to prevent the maturation of NSPs and
other serine peptidase zymogens in maturing immune cells [78,96] . Cathepsin C is also an emerging target for
the treatment of various cancers . An expert review dedicated specifically to the pharmacological targeting
[89]
of cathepsin C has been published recently . Here, we shall briefly summarize the current state-of-the-art in
[6]
the inhibition of cathepsin C.

Most cathepsin C inhibitors are peptide-based and form covalent bonds to the catalytic residue Cys234
using reactive diazomethyl-ketone, nitrile, semicarbazide, or vinyl-sulfone groups (i.e., “warheads”). Some
of the most advanced cathepsin C inhibitors to date are collected in Table 1. One of the earliest reported
cathepsin C inhibitors was Gly-Phe-CHN , a substrate analog from a series of diazomethyl-ketone
[97]
2
[98]
warhead-containing irreversible inhibitors (inactivators) of cysteine peptidases . The high reactivity of the
diazomethyl ketone group and instability in acidic media limited the use of such probes beyond in vitro
experiments, as they are metabolically unstable and sometimes have deleterious side effects, while increased
metabolic stability of such inhibitors was often accompanied by decreased inhibitory activity .
[99]

The first cathepsin C inhibitor selected as a candidate for in vivo studies was (S)-4-amino-N-(1-cyano-2-(4'-
cyano-[1,1'-biphenyl]-4-yl)ethyl)tetrahydro-2H-pyran-4-carboxamide, a reversible inhibitor with a nitrile
warhead developed by AstraZeneca (UK) and later named AZD5248 [Table 1]. The compound had pIC
[100]
50
[100]
values of 9.1 ±0.1 for purified cathepsin C in vitro and 8.1 ± 0.1 in a cell-based assay . The crystal structure
of AZD5248 bound into the active site of cathepsin C showed that it interacts with the non-primed sites of
the enzyme as well as the N-terminal part of the exclusion domain [Figure 2] . It effectively inhibited the
[100]
activation of NSPs in rats but showed aortic binding in quantitative whole-body autoradiography studies,
[101]
which could lead to potential cardiovascular toxicity. At the molecular level, aortic binding has been
associated with cross-reactivity of AZD5248 with aldehydes, resulting in cross-linking with elastin in the
[102]
aortic wall . Therefore, novel inhibitors based on AZD5248 were prepared to overcome aortic binding,
including the most advanced inhibitor to date, AZD7986 ((S)-N-((S)-1-cyano-2-(4-(3-methyl-2-oxo-2,3-
dihydrobenzo-[d]oxazol-5-yl)phenyl)ethyl)-1,4-oxazepane-2-carboxamide) . Like AZD5248, AZD7986 is
[103]
also a highly potent, reversible, and selective inhibitor of cathepsin C with pIC values of 8.4 for cathepsin C
50
in vitro and cell-based assays . It almost completely inhibited the activation of NSPs in primary CD34
+
[103]