COVID-19 is caused by inhalation of the latest coronavirus (CoV) SARS-CoV-2 into the lungs, and airwayepithelia are particularly susceptible to uptake this virus. Extensive evidence indicates that angiotensinconverting enzyme 2 (ACE2) binds to the S1 subunit of the SARS-CoV-2 Spike protein (S1), triggering selectiveproteolytic cleavage that liberates the S2 subunit. S2 undergoes extensive conformational changes to form a 6-helix bundle (6-HB) between Heptad Repeat (HR)-1 and HR-2 domains of S2, which ultimately results in thefusion of the viral particle with the cell membrane and subsequent viral entry. Based on the mechanism of viralentry, and supported by crystallography studies of the ACE2"¢S1 interface and the 6-HB complex of S2, enormousefforts are currently under way to develop peptide-based therapeutics to target both events: the interaction ofSARS-CoV-2 Spike with ACE2 receptor, and the fusion of the viral particle to the cell membrane. We havediscovered that exposure of well-differentiated, primary airway epithelial cultures to tobacco smoke for extendedperiods of time enhances ACE2 activity and increases binding of recombinant S1, which might explain theincreased susceptibility of smokers to COVID-19. The Receptor Binding Domain (RBD) in S1 is part of a highlymutable region, as revealed by the appearance of multiple highly infectious SARS-CoV-2 variants in late 2020;thus, targeting this region might not be ideal for antiviral development. In contrast, the HR regions of the S2subunit and the interaction mode of HR-1 and HR-2 domains within the 6-HB complex are highly conservedamong various CoVs, which makes it an optimal target to develop broad-spectrum antivirals. EK1 is a peptidethat. The goal of this application is to develop novel peptides that target the HR1 domain of the S2 subunit toinhibit membrane fusion and pseudovirus infection of SARS-CoV-2 as well as several other CoVs. Thesepeptides should serve as broad-spectrum CoV antivirals for the treatment of COVID-19 and subsequent COVIDs.We propose to evaluate the proteolytic stability of several peptides in the hostile environment of the lung, as themain entry way of SARS-CoV-2, including stapled and N-capped peptides with enhanced helical constraint. Wewill measure the proteolytic stability of the peptides ex vivo using human lung secretions obtained from smokersand non-smokers. We will use primary airway epithelial cells to interrogate the ability of the peptides to inhibitfusion and SARS-CoV-2 pseudovirus infection to healthy and smoke-exposed airway cultures. The efficacy ofthese peptides will be ultimately evaluated in animal models. This study will address the feasibility of helicalmimics to inhibit viral fusion and suppress viral entry into airway epithelia as a novel effective treatment againstCOVID-19.
Public Health Relevance Statement: PROJECT NARRATIVE
The virus SARS-CoV-2 causes COVID-19 by binding to a helical region of the lung surface receptor ACE2
followed by proteolytic processing and fusion to the cell membrane. Targeting viral entry by means of peptide-
mimics that block interaction of the S1 protein with ACE2 receptor or inhibit fusion of the S2 subunit to the cell
membrane offer promising therapies for the treatment of COVID-19 and other CoVs. This application is to
develop novel peptides that target SARS-CoV-2 entry that will resist degradation by enzymes secreted in the
lung; effectiveness of these peptidomimetics will be compared in healthy lung cells and in smoke-exposed cells,
which enhances ACE2 activity and viral entry, as an initial attempt to test whether these peptides could also work
in people with underlying lung conditions.
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