E-64c-Hydrazide Based Cathepsin C Inhibitors: Optimizing the Interactions with the S1'-S2' Area.

The zymogens of the neutrophil serine proteases elastase, proteinase 3, and cathepsin G are converted proteolytically into their pro-inflammatory active forms by the action of cathepsin C. The inhibition of this cysteine protease therefore is an interesting therapeutic approach for the treatment of inflammatory disorders with a high neutrophil burden such as COPD. Based on E-64c-hydrazide as lead structure, we have recently developed a covalently acting cathepsin C inhibitor using a n-butyl residue attached at the amine nitrogen of the hydrazide moiety to efficiently address the deep hydrophobic S2 pocket. To further optimize the affinity and selectivity profile of this inhibitor, the S1'-S2' area was now investigated by a combinatorial approach, showing that Nle-tryptamide is a ligand superior to the initially used Leu-isoamylamide. Using the neutrophil precursor line U937 as a cell culture model, this optimized inhibitor blocks the intracellular cathepsin C activity and thereby suppresses the activation of neutrophil elastase.

E-64c-hydrazide: a lead structure for the development of irreversible cathepsin†C inhibitors.

Cathepsin C is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k2/Ki =140±5 M(-1) s(-1)) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1'-S2' area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k2/Ki =56 000±1700 M(-1) s(-1)).