Glycosaminoglycans and Glycosaminoglycan Mimetics as Human Neutrophil Elastase Inhibitors for Cystic Fibrosis Management
Authors: Afosah Daniel Kwame , Sankaranarayanan Nehru Viji , Morla Shravan , Kumar Megh , Gangji Rahaman Navaz , Kummarapurugu Apparao , Zheng Shuo , Voynow Judith , Desai Umesh 
Published: June 2021

Cystic fibrosis (CF), a genetic disease, affects multiple organs, especially the lungs. Human neutrophil elastase (HNE), a serine protease vital for host defense mechanism, is markedly elevated in CF and plays a key role in disease progression. In fact, it is a biomarker of disease progression. Yet, there are no FDA‐approved small molecule inhibitors for HNE. The current CF therapy employs a host of symptom‐alleviating agents including DNAse to lyse mucous, hypertonic saline to aid mucous clearance, and antibiotics to combat infection. CF therapy would benefit greatly from HNE inhibitors.Previous work has shown that HNE is potently inhibited by negatively charged molecules such as heparin. Yet, heparin's powerful anticoagulant property limits its usefulness in CF. To identify selective glycosaminoglycan (GAG)‐based inhibitors of HNE, we studied the anti‐HNE activity of various GAG species of different sulfation patters, molecular weights and chain lengths. Our studies indicate that GAG sulfation patterns different from those required for anti‐coagulant property drive HNE inhibition. The results also show that sequences longer than hexasaccharides are required for anti‐HNE activity. Michaelis‐Menten kinetics suggests an allosteric mechanism of inhibition, which provides an advantage for the development of selective agents. Additionally, salt dependence studies of HNE inhibition points to the involvement of both ionic and non‐ionic interactions for HNE inhibition.Based on these results, we screened a library of 60 diverse, non‐saccharide GAG mimetics (NSGMs) against HNE, of which 10 molecules were found to be nanomolar inhibitors. Since hypertonic saline is an integral part of current CF therapy, we studied the effect of high salt levels on these inhibitors and identified six NSGMs that retained excellent potency, probably arising from considerable specific, non‐ionic interactions in binding to HNE. Studies on inhibition of HNE degradation of macromolecular substrate elastin led to further winnowing of inhibitors to two promising NSGMs. Finally, studies on a panel of serine proteases and coagulation assays led to the selection of a tetrasulfated diflavonoid NSGM for further testing in sputum from CF patients. This agent showed a dose‐dependent decrease in HNE activity of CF sputum when used in combination with DNAse. These studies indicate that GAG‐based inhibition of HNE is a promising avenue for developing anti‐CF agents. More specifically, the results suggest that a tetra‐sulfated diflavonoid NSGM could add value to the trio of DNAse, hypertonic saline and antibiotics used currently in the clinic.Support or Funding InformationThis work is funded by NHLBI grant 1P01HL107152 to Dr. Umesh R. Desai.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.