Glycosaminoglycans as Multifunctional Anti-Elastase and Anti-Inflammatory Drugs in Cystic Fibrosis Lung Disease

Abstract

Neutrophil elastase (NE) is a major protease in the airways of patients with cystic fibrosis (CF) that activates airway inflammation by several mechanisms. NE stimulates epithelial toll like receptors (TLR) resulting in cytokine upregulation and release, upregulates MUC5AC, a major airway mucin, degrades both phagocytic receptors and opsonins resulting in both neutrophil and macrophage phagocytic failure, generates oxidative stress via extracellular generation and uptake of heme free iron, and activates other proteases. Altogether, these mechanisms create a significant inflammatory challenge that impairs innate immune function and results in airway remodeling. Currently, a major gap in our therapeutic approach to CF lung disease is the lack of an effective therapeutic strategy targeting active NE and its downstream pro-inflammatory sequelae. Polysulfated glycosaminoglycans (GAGs) are potent anti-elastase drugs that have additional anti-inflammatory properties. Heparin is a prototype of a glycosaminoglycan with both anti-elastase and anti-inflammatory properties. Heparin inhibits NE in an allosteric manner with high potency. Heparin also inhibits cathepsin G, blocks P-selectin and L-selectin, hinders ligand binding to the receptor for advanced glycation endproducts, and impedes histone acetyltransferase activity which dampens cytokine transcription and High Mobility Group Box 1 release. Furthermore, nebulized heparin treatment improves outcomes for patients with chronic obstructive pulmonary disease (COPD), asthma, acute lung injury and smoke inhalation. However, the anticoagulant activity of heparin is a potential contraindication for this therapy to be developed for CF lung disease. Therefore, modified heparins and other GAGs are being developed that retain the anti-elastase and anti-inflammatory qualities of heparin with minimal to no anticoagulant activity. The modified heparin, 2-O, 3-O desulfated heparin (ODSH), maintains anti-elastase and anti-inflammatory activities in vitro and in vivo, and has little residual anticoagulant activity. Heparan sulfate with O-sulfate residues but not N-sulfate residues blocks allergic asthmatic inflammation in a murine model. Polysulfated hyaluronic acid abrogates allergen- triggered rhinosinusitis in a murine model. Finally, nonsaccharide glycosaminoglycan mimetics with specific sulfate modifications can be designed to inhibit NE activity. Altogether, these novel GAGs or GAG mimetics hold significant promise to address the unmet need for inhaled anti-elastase and anti-inflammatory therapy for patients with CF.

Keywords: High Mobility Group Box 1; cystic fibrosis; glycosaminoglycans; heparin; hyaluronic acid; neutrophil elastase.

Figure 1

ODSH localization and function in a macrophage cell line. ODSH is taken up by a mouse macrophage cell line (RAW264.7) into the cytoplasm within 2 h and into the nucleus by 24 h (Zheng et al., 2017) (A). ODSH has anti-NE activity and blocks HMGB1-RAGE interaction in the extracellular domain, and inhibits p300 lysine acetyltransferase activity in the nucleus (A). ODSH inhibits NE activity by binding to an allosteric inhibitory site (Kummarapurugu et al., 2018), and ODSH inhibits p300 enzyme activity by binding to the acetyl-CoA binding site in the catalytic domain (Zheng et al., 2017) (B). In contrast, ODSH binds to the loop connecting the A-box and B-box of HMGB1, blocking interaction with heparan sulfate proteoglycans required for HMGB1 ligation of the RAGE receptor (Xu et al., 2011) (B). Amino acid residues required for ODSH or heparin inhibitory activity are shown (Red Box).

Figure 2

Heparin/ODSH interrupts cell- cell interactions and ligand-receptor binding to block pro-inflammatory pathways. Heparin/ODSH oligosaccharides bind to P- and L-selectins and block neutrophil adhesion and chemotaxis (Nelson et al., 1993; Rao et al., 2010). Heparin inhibits CXCL8/IL-8 and other ELR (Glu Leu Arg)-CXC chemokines from binding to G-protein coupled receptors CXCR1 and CXCR2 (Rajarathnam KaD, 2020). Heparin competes with HSPG for binding to microbial proteins which prevents bacterial or viral-epithelial adhesion and invasion (Rostand and Esko, 1997). Heparin/ODSH bind to HMGB1 and S100A9 and interrupt RAGE ligation (Rao et al., 2010). HSPG, heparan sulfate proteoglycan; NTHi, non-typeable H. influenza; S100A9, calgranulin; SARS-CoV, Severe acute respiratory syndrome- corona virus.