SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α₁-antitrypsin (α₁AT), a highly abundant circulating serine protease inhibitor. Here, we report that α₁AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α₁AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α₁AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α₁AT-containing drugs has prospects for the therapy of COVID-19.

Membrane fusion triggered by Ca²⁺ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes^1–4. For neurotransmitter release, the Ca²⁺ sensitivity is introduced by interactions between the Ca²⁺ sensor synaptotagmin and the SNARE complex⁵, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion⁶. Disruption of Ca²⁺-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^7–11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca²⁺-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca²⁺-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 Ca²⁺-triggered fusion at physiological Ca²⁺ 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 Ca²⁺-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Vesicular P2X₄ receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X₄ receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X₄ receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X₄ receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X₄ by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X₄) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X₄), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion.

The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells secrete pulmonary surfactant, regulate the alveolar surface liquid (ASL) volume and, together with resident and infiltrating immune cells, provide a powerful host-defense system against a multitude of particles, microbes and toxicants. It is well established that all of these cells express purinergic P2 receptors and that purinergic signaling plays important roles in maintaining alveolar homeostasis. Therefore, it is not surprising that purinergic signaling also contributes to development and progression of severe pathological conditions like pulmonary inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis. Within this review we focus on the role of P2 purinergic signaling in the distal lung in health and disease. We recapitulate the expression of P2 receptors within the cells in the alveoli, the possible sources of ATP (adenosine triphosphate) within alveoli and the contribution of purinergic signaling to regulation of surfactant secretion, ASL volume and composition, as well as immune homeostasis. Finally, we summarize current knowledge of the role for P2 signaling in infectious pneumonia, ALI/ARDS and idiopathic pulmonary fibrosis (IPF).