CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells, and its pulmonary levels correlate with viral clearance in critical COVID-19 patients.

Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage, and cytokine storm. One unexplored component in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients and restored to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analog SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatments with CGRP and/or SAX are enough to block SARS-CoV-2 productive infection of Calu-3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of both SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients by directly inhibiting SARS-CoV-2 propagation. Hence, CGRP-based interventions could be harnessed for management of COVID-19.IMPORTANCEThe neuropeptide CGRP is highly abundant in the airways. Due to its immunomodulatory, vasodilatory, and anti-viral functions, CGRP could affect multiple aspects of COVID-19-related pulmonary pathophysiology. Yet, the interplay between CGRP and SARS-CoV-2 during COVID-19 remains elusive. Herein, we show that pulmonary levels of CGRP are increased in critical COVID-19 patients, at an early stage of their disease when patients are SARS-CoV-2-positive. Upon subsequent viral clearance, CGRP levels are restored to baseline in SARS-CoV-2-negative patients. We further show that pre- and post-infection treatments with CGRP directly inhibit infection of Calu-3 bronchial epithelial cells with SARS -CoV-2, via activation of the CGRP receptor leading to decreased expression of both SARS-CoV-2 entry receptors. Together, we propose that increased pulmonary CGRP is beneficial in COVID-19, as CGRP-mediated inhibition of SARS-CoV-2 infection could contribute to viral clearance in critical COVID-19 patients. Accordingly, CGRP-based formulations could be useful for COVID-19 management.

Selection of Bis-Indolyl Pyridines and Triphenylamines as New Inhibitors of SARS-CoV-2 Cellular Entry by Modulating the Spike Protein/ACE2 Interfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC50) between 0.1 and 0.5 µM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.

Short-term exposure to cigarette smoke upregulates cathepsin S and alters expression of tight junction ZO-1.

A long-term exposure to cigarette smoke (CS) alters the integrity of airway epithelial barrier, contributes to lung dysfunction, and elicits the expression and activity of lung cathepsin S (CatS), a cysteine protease that participates in the remodeling of connective tissue and cell junctions. Here, we observed that a short-term (4 days) exposure of mice to CS increased the expression and activity of CatS, while the expression level of zonula occludens 1 (ZO-1), an epithelial tight junction protein that stabilizes barrier assembly, was reduced in lung tissue lysates. Present data support that proteolytically active CatS may contribute to the defect of ZO-1 in CS-exposed mice.

Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly.

Considerable progress has been made in understanding the molecular host-virus battlefield during SARS-CoV-2 infection. Nevertheless, the assembly and egress of newly formed virions are less understood. To identify host proteins involved in viral morphogenesis, we characterize the proteome of SARS-CoV-2 virions produced from A549-ACE2 and Calu-3 cells, isolated via ultracentrifugation on sucrose cushion or by ACE-2 affinity capture. Bioinformatic analysis unveils 92 SARS-CoV-2 virion-associated host factors, providing a valuable resource to better understand the molecular environment of virion production. We reveal that G3BP1 and G3BP2 (G3BP1/2), two major stress granule nucleators, are embedded within virions and unexpectedly favor virion production. Furthermore, we show that G3BP1/2 participate in the formation of cytoplasmic membrane vesicles, that are likely virion assembly sites, consistent with a proviral role of G3BP1/2 in SARS-CoV-2 dissemination. Altogether, these findings provide new insights into host factors required for SARS-CoV-2 assembly with potential implications for future therapeutic targeting.