In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Abstract

Inhibition of cathepsin C, a dipeptidyl peptidase that activates many serine proteases, represents an attractive therapeutic strategy for inflammatory diseases with a high neutrophil burden. We recently showed the feasibility of blocking the activation of neutrophil elastase, cathepsin G, and proteinase-3 with a single cathepsin C selective inhibitor in cultured cells. Here we measured the fractional inhibition of cathepsin C that is required for blockade of downstream serine protease processing, in cell-based assays and in vivo. Using a radiolabeled active site probe and U937 cells, a 50% reduction of cathepsin G processing required ϳ50% of cathepsin C active sites to be occupied by an inhibitor. In EcoM-G cells, inhibition of 50% of neutrophil elastase activity required ϳ80% occupancy. Both of these serine proteases were fully inhibited at full cathepsin C active site occupancy, whereas granzyme B processing in TALL-104 cells was partially inhibited, despite complete occupancy. In vivo, leukocytes from cathepsin C ϩ/Ϫ mice exhibited comparable levels of neutrophil elastase activity to wild-type animals, even though their cathepsin C activity was reduced by half. The long-term administration of a cathepsin C inhibitor to rats, at doses that resulted in the nearly complete blockade of cathepsin C active sites in bone marrow, caused significant reductions of neutrophil elastase, cathepsin G and proteinase-3 activities. Our results demonstrate that the inhibition of cathepsin C leads to a decrease of activity of multiple serine proteases involved in inflammation but also suggest that high fractional inhibition is necessary to reach therapeutically significant effects.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

“…For this therapeutic application, CatC activity must be reduced by Ͼ95%. Our data support the conclusions of Méthot et al (25,26) who reported that very high fractional inhibition of the CatC activity by chronic administration of a CatC inhibitor is required to prevent the activation of NSPs in vivo. CatH has been shown to serve as a backup for CatC-mediated granzyme B activation in cytotoxic lymphocytes (38).…”

“…In the past years dipeptidyl nitriles have attracted great attention due to their strong inhibitory activity against human CatC (23,24). Inactivation of CatC in the bone marrow of rats by treating them with a reversible cyclopropyl nitrile inhibitor for 2 weeks at doses that resulted in the nearly complete inhibition of CatC only partially prevents activation of NSPs (25). Thus, a continuous very high degree of CatC inhibition is required to effectively block the maturation of downstream elastase-like NSPs in vivo (25,26).…”

“…Inactivation of CatC in the bone marrow of rats by treating them with a reversible cyclopropyl nitrile inhibitor for 2 weeks at doses that resulted in the nearly complete inhibition of CatC only partially prevents activation of NSPs (25). Thus, a continuous very high degree of CatC inhibition is required to effectively block the maturation of downstream elastase-like NSPs in vivo (25,26). These genetic and pharmacological data support the hypothesis that CatC is a therapeutic target for the treatment of several inflammatory and autoimmune diseases affecting the respiratory systems (23,27).…”

Diverse regulatory roles for lysosomal proteases in the immune response

“…These data indicate that cathepsin C may be an attractive drug target to control multiple inflammatory serine proteases. Nonetheless, it appears that efficient and sustained inhibition of the enzyme will be required to fully quench neutrophil granule protease activity [63]. Cathepsin C may itself be regulated by cystatin F. Cystatins are a family of proteinaceous cysteine protease inhibitors, some of which bind very tightly to cathepsin active sites [64].…”

Discovery of Novel Cyanamide-Based Inhibitors of Cathepsin C

“…3 There is increasing evidence that cathepsin C plays a key role in a variety of diseases, including sepsis, 4 arthritis, 5 and other inflammatory disorders. [6][7][8][9][10][11][12] From knockout mice studies, it appears that cathepsin C is coexpressed and acts as a physiological activator of the neutrophil-derived serine proteases: neutrophil elastase, cathepsin G and proteinase 3,5 the mast cell chymase and trypase, 13 and the lymphocytes derived granzymes A and B.…”