An uncontrolled activity of neutrophil serine proteases (NSPs; elastase, proteinase 3 and cathepsin G) contributes to chronic inflammatory pulmonary diseases. Cathepsin C (CatC), also known as dipeptidyl peptidase I (DPPI), executes the activation of NSPs and represents a promising pharmacological target in NSP-mediated diseases. Results from the Phase 3 ASPEN study using brensocatib, an active site targeting inhibitor, validate CatC as a pharmacological target in non-cystic fibrosis bronchiectasis. CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages (Figure 1). Pharmacological inhibition of CatC can be achieved either using chemical inhibitors interacting with the mature, functional protease (direct inhibition) or by impairing the activation of its proform by targeting proCatC-converting protease(s) (indirect inhibition). In this study, we investigated the consequences of indirect pharmacological inhibition of CatC on the activation of NSPs.

CatC is maturated by CatL-like proteases (CatS, CatL, CatF, CatK and CatV) in vitro. CatL-like proteases that could maturate proCatC were investigated by proteomic approaches in neutrophilic precursor cells. Human CD₃₄ ⁺ cells were differentiated in neutrophils with or without a nitrile CatS inhibitor, IcatS_#54. CatC and NSPs were analysed in neutrophil lysates by western blotting and kinetic assays. Proteolytic pathways involved in the activation of NSPs were studied in neutrophils derived from CatC knock-out (Ctsc^−/−) or CatS knock-out (Ctss^−/−) mice.

We show that CatS, a potent elastinolytic enzyme, is the major druggable converting protease involved in the maturation of human neutrophil proCatC. Pharmacological targeting of intracellular CatS using IcatS_#54 almost completely blocked CatC maturation in CD₃₄ ⁺-derived neutrophils and resulted in inactivation and elimination of NSPs (∼70 %). However, CatS is not the major protease responsible for CatC maturation in rodent. Characterisation of proteolytic cascades involved in the activation of macaque NSPs is under investigation.

We established a proof of concept for the indirect inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP activation using a CatS inhibitor, alone or in combination with a CatC, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases.