Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides.

Membrane fusion triggered by Ca is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes. For neurotransmitter release, the Ca sensitivity is introduced by interactions between the Ca sensor synaptotagmin and the SNARE complex, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion. Disruption of Ca-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca-triggered fusion at physiological Ca concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.