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A1210 - The ENaC Regulatory Peptide SPX-101 Is Resistant to Proteolytic Degradation in Diseased Sputum
Author Block: D. Scott, T. Stuhlmiller, B. Wu, J. Sesma; Spyryx Biosciences, Durham, NC, United States.
Introduction: Mucus dehydration and decreased mucociliary clearance are hallmarks of multiple pulmonary diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and non-CF bronchiectasis (NCFBE). Airway hydration is controlled by the epithelial sodium channel (ENaC) and its natural allosteric regulator short palate lung and nasal clone 1 (SPLUNC1). It has been reported that SPLUNC1 is absent in the sputum of CF and COPD patients and that the protein is degraded by neutrophil elastase. This suggests that loss of SPLUNC1 could play a major role in mucus dehydration associated with these diseases. We have developed a novel ENaC regulatory peptide, SPX-101, that can replace SPLUNC1 function. This work investigates the abundance of SPLUNC1 in sputum from healthy and diseased subjects, the stability of SPLUNC1 and SPX-101 in sputum from diseased patients, and the functionality of SPX-101 after exposure to diseased sputum. Methods: SPLUNC1 abundance was determined by western blot analysis. Stability of SPLUNC1 in sputum was determined by western blot while stability of SPX-101 was assessed by HPLC. The function of SPX-101 after exposure to sputum samples was determined by analysis of ENaC surface density and airway surface liquid height in air liquid interface cultured human bronchial epithelial cells. Results: SPLUNC1 protein was detectable in the sputum of all healthy subjects, in one of 10 CF subjects, in five of 11 COPD subjects, and in zero of six NCFBE subjects. Recombinant SPLUNC1 was rapidly degraded in samples absent of the endogenous protein. This degradation could be prevented by heat inactivation of the sputum samples suggesting that active proteases were responsible. Incubation of sputum samples with sivelestat, a neutrophil elastase inhibitor, was not sufficient to prevent SPLUNC1 degradation implicating additional proteases in the breakdown of the protein. Indeed, multiple disease-associated proteases were found to degrade SPLUNC1. In contrast, SPX-101 was stable in these same sputum samples and proteases. Moreover, SPX-101 which had been exposed to sputum from diseased patients or individual proteases retained normal ENaC regulatory function. Conclusions: ­SPLUNC1 protein is absent in the majority of sputum samples from patients with disease characterized by mucus dehydration and decreased mucociliary clearance. SPX-101, a therapeutically-optimized peptide which mimics SPLUNC1’s ENaC regulatory function, is stable in sputum samples and retains pharmacological activity thereafter.