Sulfonated Nonsaccharide Heparin Mimetics Are Potent and Noncompetitive Inhibitors of Human Neutrophil Elastase

Abstract

Human neutrophil elastase (HNE) is a serine protease that plays vital roles in inflammation, innate immune response, and tissue remodeling processes. HNE has been actively pursued as a drug target, particularly for the treatment of cardiopulmonary diseases. Although thousands of molecules have been reported to inhibit HNE, yet very few are being evaluated in early clinical trials, with sivelestat as the only approved HNE inhibitor. We report here a novel chemotype of sulfonated nonsaccharide heparin mimetics as potent and noncompetitive inhibitors of HNE. Using a chromogenic substrate hydrolysis assay, 14 sulfonated nonsaccharide heparin mimetics were tested for their inhibitory activity against HNE. Only 12 molecules inhibited HNE with IC50 values of 0.22–88.3 μM. The inhibition of HNE by these molecules was salt-dependent. Interestingly, a specific hexa-sulfonated molecule inhibited HNE with an IC50 value of 0.22 μM via noncompetitive mechanism, as demonstrated by Michaelis–Menten kinetics. The hexa-sulfonated derivative demonstrated at least 455-, 221-, 1590-, 21-, and 381-fold selectivity indices over other heparin-binding coagulation proteins including factors IIa, Xa, IXa, XIa, and FXIIIa, respectively. At the highest concentrations tested, the molecule also did not significantly inhibit other serine proteases of plasmin, trypsin, and chymotrypsin. Further supporting its selectivity, the molecule did not show heparin-like effects on clotting times of human plasma. The molecule also did not affect the proliferation of three cell lines at a concentration as high as 10 μM. Interestingly, the hexa-sulfonated molecule also inhibited cathepsin G with an IC50 value of 0.57 μM alluding to a dual anti-inflammatory action. A computational approach was exploited to identify putative binding site(s) for this novel class of HNE inhibitors. Overall, the reported hexa-sulfonated nonsaccharide heparin mimetic serves as a new platform to develop potent, selective, and noncompetitive inhibitors of HNE for therapeutic purposes.

Lignosulfonic Acid Sodium Is a Noncompetitive Inhibitor of Human Factor XIa

“…We also evaluated LSAS inhibitory potential against other proteases including plasmin, trypsin, chymotrypsin, human neutrophil elastase (HNE), and cathepsin G using the corresponding chromogenic substrate hydrolysis assays under near physiologic conditions, as reported in our earlier studies [ 7 , 8 , 9 , 11 , 12 , 13 , 14 , 15 ]. In these assays, the inhibition potential was determined by spectrophotometric measurement of the residual protease activity in the presence of varying concentrations of LSAS ( Figure 3 A,B).…”

“…Furthermore, targeting FXI(a)/FXII(a) axis by therapeutic tools has been demonstrated to prevent systemic inflammation, coagulopathy, and mortality in experimental sepsis [ 40 , 41 ]. Interestingly, we found in this study that LSAS inhibits HNE, an inflammatory serine protease the overactivity of which contributes to a host of cardiopulmonary diseases [ 13 ], with an IC 50 value of 4.7 ± 0.2 µg/mL ( Table 2 ). Furthermore, we also found that LSAS inhibits another inflammatory protease, i.e., cathepsin G [ 42 ] with an IC 50 value of 0.73 ± 0.11 µg/mL ( Table 2 ).…”

“…The inhibition potential of LSAS against plasmin, trypsin, chymotrypsin, and human neutrophil elastase (HNE) was also studied using the corresponding chromogenic tripeptide substrate hydrolysis assays as documented in our previous reports [ 7 , 8 , 9 , 11 , 12 , 13 ]. Briefly, to each well of a 96-well microplate containing 185 μL (trypsin, chymotrypsin, and HNE) or 85 µL (plasmin) of 20–50 mM Tris-HCl buffer, pH 7.4, containing, 0.02% Tween80, 0.1% PEG8000, and 100–150 mM NaCl at 37 °C was added 5 μL of LSAS (0–5.0 mg/mL in the well) (or the vehicle) and 5 μL of the enzyme.…”

Sulfonated non‐saccharide molecules and human factor XIa: Enzyme inhibition and computational studies

“…Molecules 1 – 5 and 11 were obtained from Sigma‐Aldrich and Fisher Scientific. Other molecules were obtained following modified reported synthetic protocols (Al‐Horani et al, 2021; Kassack et al, 2004; McCain et al, 2004; Trapp et al, 2007). For instance, the pure sodium salts were produced by SP Sephadex‐Na cation exchange chromatography and then by Sephadex G10 size exclusion chromatography.…”

“…The inhibition potential of several sulfonated molecules against thrombin, FXa, FIXa, plasmin, trypsin, chymotrypsin, cathepsin G, HNE, and proteinase 3 was also investigated using previously reported chromogenic substrate hydrolysis assays (Al‐Horani, Abdelfadiel, et al, 2019; Al‐Horani, Clemons, et al, 2019; Al‐Horani et al, 2015, 2021; Kar et al, 2020, 2021). For example, to each well of a 96‐well microplate containing 85 μl or 185 μl of 20–50 mM Tris‐HCl buffer, pH 7.4, containing 100–150 mM NaCl, 0.1% PEG8000, and 0.02% Tween80 at either 25°C or 37°C was added 5 μl of the inhibitor (or high pure water) and 5 μl of the enzyme.…”