The ENaC‐targeted Therapeutic Peptide SPX‐101 is Resistant to Proteolytic Degradation in Diseased Sputum

Abstract

IntroductionPulmonary diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and non‐CF bronchiectasis (NCFBE) are characterized by mucus dehydration and decreased mucociliary clearance. The epithelial sodium channel (ENaC) provides the primary driving force for water movement across airway epithelia. ENaC surface density is negatively regulated by short palate lung and nasal clone 1 (SPLUNC‐1), a protein secreted by airway epithelia. SPLUNC‐1 is among the most‐abundant proteins in airway surface liquid (ASL), but SPLUNC‐1 is reported to be reduced or absent in sputum from individuals with COPD and CF. This suggests the excessive airway dehydration characteristic of these diseases could be led by the loss of SPLUNC‐1‐mediated ENaC inhibition. We have developed an optimized therapeutic peptide, SPX‐101, that replaces the ENaC internalization function of SPLUNC‐1. In animal models of CF, SPX‐101 prolongs survival and enhances mucociliary clearance. The objective of this study was to determine the abundance of SPLUNC‐1 in sputum derived from individuals with COPD, CF and NCFBE as compared to healthy individuals, and to determine the stability and functionality of SPLUNC‐1 and SPX‐101 after exposure to diseased sputum.MethodsSPLUNC‐1 protein levels in COPD, CF, and NCFBE sputum were determined by western blot. After exposure to sputum at various time points, stability of exogenous SPLUNC‐1 protein was determined by western blot and stability of SPX‐101 by HPLC. SPX‐101 functionality after exposure to sputum was determined by its ability to internalize ENaC and increase ASL height in human bronchial epithelial cells cultured at air‐liquid interface.ResultsSPLUNC‐1 protein was detectable in the sputum of all healthy subjects, in five of 11 COPD subjects, one of 10 CF subjects, and in zero of six NCFBE subjects. Exogenous purified SPLUNC‐1 protein was rapidly degraded in samples absent of the endogenous protein. Degradation was prevented by heat inactivation of the sputum samples suggesting the contribution of active proteases. Inhibition of neutrophil elastase with sivelestat was not sufficient to prevent SPLUNC‐1 cleavage, indicating additional proteases present in diseased sputum contribute to SPLUNC‐1 degradation. In agreement, multiple disease‐associated proteases were found to degrade SPLUNC‐1. In contrast, SPX‐101 was stable in these same sputum samples and proteases. Furthermore, SPX‐101 retained normal ENaC regulatory functions after exposure to sputum from diseased patients or incubation with purified proteases.ConclusionsSPLUNC‐1 protein was undetectable in the majority of sputum samples from patients with COPD, CF, and NCFBE. SPX‐101, a therapeutically‐optimized peptide which retains the negative ENaC regulatory function of SPLUNC‐1, is stable in sputum samples and retains pharmacological activity where SPLUNC‐1 is absent. This suggests SPX‐101 could benefit individuals where loss of SPLUNC‐1 function is a hallmark of disease manifestation.Support or Funding InformationThis work was funded in part by a grant from the Cystic Fibrosis Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.