N-Arylacyl O-sulfonated aminoglycosides as novel inhibitors of human neutrophil elastase, cathepsin G and proteinase 3

Abstract

The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N-arylacyl O-sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated.

Keywords: glycosaminoglycans; heparin mimics; inflammatory lung disease; neutrophil serine proteases; protease inhibitors.

Fig. 1.

Structures and degree of sulfation (DS) of N-arylacyl O-sulfonated aminoglycosides used in this study. The three aminoglycoside neomycin, kanamycin and apramycin are N-substituted with three different arylacyl groups and per-O-sulfonated, affording the panel of nine derivatives: N-carbobenzyloxy O-sulfonated neomycin (NeoCbz, DS = 7), N-phenylacetyl O-sulfonated neomycin (NeoPhA, DS = 6.8), N-benzoyl O-sulfonated neomycin (NeoBz, DS = 7), N-carbobenzyloxy O-sulfonated kanamycin (KanCbz, DS = 6), N-phenylacetyl O-sulfonated kanamycin (KanPhA, DS = 5.8), N-benzoyl O-sulfonated kanamycin (KanBz, DS = 5.1), N-carbobenzyloxy O-sulfonated apramycin (AprCbz, DS = 6), N-phenylacetyl O-sulfonated apramycin (AprPhA, DS = 6), N-benzoyl O-sulfonated apramycin (AprBz, DS = 6). Complete synthesis and characterization of panel of compounds is described in Supplementary data. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 2.

NSP inhibition by N-arylacyl O-sulfonated aminoglycosides. (A–C) Dose-dependent inhibition of HNE by neomycin, kanamycin and apramycin derivatives, respectively; (D–F) dose-dependent inhibition of CatG by neomycin, kanamycin and apramycin derivatives, respectively; (G–I) dose-dependent inhibition of Pr3 by neomycin, kanamycin and apramycin derivatives, respectively. Absorbance of p-nitroaniline released from a protease specific chromogenic substrate was measured at 405 nm and normalized to enzyme only control. Nonlinear regression curve fitting was performed to obtain IC₅₀ values summarized in Table I. Data are presented as mean ± SE (n = 3). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 3.

Mode of CatG inhibition. Michaelis–Menten plot and double reciprocal Lineweaver-Burk plot (inset) of (A) KanCbz and (B) NeoCbz inhibition of CatG. The concentration of CatG chromogenic substrate was varied at four different inhibitor concentrations (0, 0.5[IC₅₀], [IC₅₀], 2[IC₅₀] and 4[IC₅₀]). Both compounds were partial mixed inhibitors of CatG.

Fig. 4.

Protection against protease-mediated cell detachment. A549 lung epithelial cells were exposed to (A) HNE (50 nM), (B) CatG (250 nM) or (C) Pr3 (50 nM) in the presence or absence of decreasing concentrations of KanCbz. The NSPs were pre-incubated with KanCbz (250, 2.50 and 0.025 μM) for 30 min on ice before addition to confluent A549 cells. After 24 h lung epithelial cell nuclei were stained with Hoechst 33342 and cells were imaged using Operetta High Content Imaging System. Only one representative field for each experiment is shown for clarity. At 250 μM KanCbz inhibits HNE-, CatG- and Pr3-induced cell detachment. At lower concentrations, this protection is lost and protease-mediated cell detachment is once again observed. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 5.

Quantification of NSP-mediated cell detachment in the presence of two lead compounds. A549 lung epithelial cells were exposed to each NSP (50 nM HNE, 250 nM CatG or 50 nM Pr3) in the presence of decreasing concentrations of KanCbz (A–C) or NeoCbz (D–F). After 24 h lung epithelial cell nuclei were stained with Hoechst 33342 and cells were imaged and counted using an Operetta High Content Imaging System and Harmony Analysis Software, respectively. At the highest concentration, both KanCbz and NeoCbz protected cells against protease-mediated cell detachment. Data are presented as mean + SE, **P < 0.01, ***P < 0.001 ****P < 0.0001 when compared with protease-treated cells (n = 3 from three experiments each done in triplicate). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 6.

Comparison of crystal structures of the three NSPs. Ribbon representation of (A) HNE (PDB: 1PPF) (B) CatG (PDB: 1CGH) and (C) Pr3 (PDB: 1FUJ). Arginine and lysine side chains are shown on the surface of the structures and side chains of the catalytic triad residues (His57, Asp102 and Ser195) in the center of each structure. This figure is available in black and white in print and in color at Glycobiology online.