Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

Abstract

The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+ stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly α-helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle content-mixing assay with reconstituted neuronal SNAREs and synaptotagmin-1 or with reconstituted airway SNAREs and synaptotagmin-2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptide that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion that is a major cause of airway obstruction in the pathophysiology of asthma and cystic fibrosis.

Discovery of a drug to treat airway mucus hypersecretion

“…Four of eleven peptides with the highest degree of α‐helicity inhibited calcium‐triggered fusion, and one of the most efficacious of these (termed SP9) was also tested in a reconstituted airway secretory system where it again specifically inhibited calcium‐triggered secretion. 19 Besides stabilizing peptide structure, the staples also reduced undesirable side effects by preventing peptide interactions with the ternary SNARE complex required for baseline mucin secretion and constitutive non‐mucin secretion. Next, SP9 was conjugated to cell penetrating peptides and added to cultured human airway epithelial cells.…”

“…Next, a series of stapled peptides based on the region of SNAP‐25 known to interact with Syt1 were screened for their ability to inhibit a fusion of reconstituted synaptic vesicles with reconstituted neuronal plasma membranes. Four of eleven peptides with the highest degree of α‐helicity inhibited calcium‐triggered fusion, and one of the most efficacious of these (termed SP9) was also tested in a reconstituted airway secretory system where it again specifically inhibited calcium‐triggered secretion 19 . Besides stabilizing peptide structure, the staples also reduced undesirable side effects by preventing peptide interactions with the ternary SNARE complex required for baseline mucin secretion and constitutive non‐mucin secretion.…”

“…18 These four lines of investigation came together in collaborative work that has been recently published. 11,19 First, Syt2 was validated as a therapeutic target to treat mucus hypersecretion by conditionally deleting it in airway epithelial cells of mice. Deletant mice showed marked reductions in stimulated mucin secretion and lumenal mucus occlusion in a model of allergic asthma, without an impairment of homeostatic baseline mucin secretion.…”

“…These four lines of investigation came together in collaborative work that has been recently published 11,19 . First, Syt2 was validated as a therapeutic target to treat mucus hypersecretion by conditionally deleting it in airway epithelial cells of mice.…”