Inhibition of Airway Epithelial Snare/Synaptotagmin Mediated Membrane Fusion by Hydrocarbon-Stapled Peptides

Abstract

Airway mucin secretion involves membrane fusion of secretory granules that is mediated by SNAREs (syntaxin-3, SNAP-23, VAMP8), synaptotagmin-2, and other proteins. Mucus hypersecretion is a major cause of airway obstruction in the pathophysiology of asthma and cystic fibrosis. Deletion of synaptotagmin-2 protects mice against airway obstruction in a model of allergic asthma indicating the rationality of synaptotagmin-2 as a target for drug design. The so-called primary interface between the neuronal SNARE complex and synaptotagmin-1 is important for neurotransmitter release (Zhou et al., 2015), and it is also conserved for airway epithelial SNAREs and synaptotagmin-2. We designed a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface. The most α-helical stapled peptide had an affinity to the synaptotagmin-1 C2B domain that is similar to the affinity between the SNARE complex and the C2B domain. A subset of four stapled peptides, which were α-helical as assessed by circular dichroism, inhibit Ca2+-triggered fusion in a single vesicle fusion assay with reconstituted airway SNAREs and synaptotagmin-2. Inclusion of both Munc13-2 and Munc18-2 in the single vesicle fusion assay, the stapled peptide SP9 strongly suppress the Ca2+-triggered fusion even at the physiological Ca2+ concentrations. Moreover, cell penetrating peptide adjunct stapled peptide can be efficiently delivered into cultured epithelial cells and markedly reduced stimulated mucin secretion. Taken together, hydrocarbon-stapled peptide that interfere with the synaptotagmin-2/SNARE complex primary interface may allow modulation of secretory pathways.