Indole-3-carbinol in vitro antiviral activity against SARS-Cov-2 virus and in vivo toxicity.

The effects of indole-3-carbinol (I3C) compound have been described deeply as antitumor drug in multiple cancers. Herein, I3C compound was tested for toxicity and antiviral activity against SARS-CoV-2 infection. Antiviral activity was assessed in vitro in both in VeroE6 cell line and human Lung Organoids (hLORGs) where I3C exhibited a direct anti-SARS-CoV-2 replication activity with an antiviral effect and a modulation of the expression of genes implicated in innate immunity and inflammatory response was observed at 16.67 µM. Importantly, we further show the I3C is also effective against the SARS-CoV-2 Omicron variant. In mouse model, instead, we assessed possible toxicity effects of I3C through two different routes of administration: intragastrically (i.g.) and intraperitoneally (i.p.). The LD50 (lethal dose 50%) values in mice were estimated to be: 1410 and 1759 mg/kg i.g.; while estimated values for i.p. administration were: 444.5 mg/kg and 375 mg/kg in male and female mice, respectively. Below these values, I3C (in particular at 550 mg/kg for i.g. and 250 mg/kg for i.p.) induces neither death, nor abnormal toxic symptoms as well as no histopathological lesions of the tissues analysed. These tolerated doses are much higher than those already proven effective in pre-clinical cancer models and in vitro experiments. In conclusion, I3C exhibits a significant antiviral activity, and no toxicity effects were recorded for this compound at the indicated doses, characterizing it as a safe and potential antiviral compound. The results presented in this study could provide experimental pre-clinical data necessary for the start of human clinical trials with I3C for the treatment of SARS-CoV-2 and beyond.

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids.

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19.

Peptide Platform as a Powerful Tool in the Fight against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing over 195 million infections and more than 4 million fatalities as of July 2021.To date, it has been demonstrated that a number of mutations in the spike glycoprotein (S protein) of SARS-CoV-2 variants of concern abrogate or reduce the neutralization potency of several therapeutic antibodies and vaccine-elicited antibodies. Therefore, the development of additional vaccine platforms with improved supply and logistic profile remains a pressing need. In this work, we have validated the applicability of a peptide-based strategy focused on a preventive as well as a therapeutic purpose. On the basis of the involvement of the dipeptidyl peptidase 4 (DPP4), in addition to the angiotensin converting enzyme 2 (ACE2) receptor in the mechanism of virus entry, we analyzed peptides bearing DPP4 sequences by protein-protein docking and assessed their ability to block pseudovirus infection in vitro. In parallel, we have selected and synthetized peptide sequences located within the highly conserved receptor-binding domain (RBD) of the S protein, and we found that RBD-based vaccines could better promote elicitation of high titers of neutralizing antibodies specific against the regions of interest, as confirmed by immunoinformatic methodologies and in vivo studies. These findings unveil a key antigenic site targeted by broadly neutralizing antibodies and pave the way to the design of pan-coronavirus vaccines.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

Inhibition of HECT E3 ligases as potential therapy for COVID-19.

SARS-CoV-2 is responsible for the ongoing world-wide pandemic which has already taken more than two million lives. Effective treatments are urgently needed. The enzymatic activity of the HECT-E3 ligase family members has been implicated in the cell egression phase of deadly RNA viruses such as Ebola through direct interaction of its VP40 Protein. Here we report that HECT-E3 ligase family members such as NEDD4 and WWP1 interact with and ubiquitylate the SARS-CoV-2 Spike protein. Furthermore, we find that HECT family members are overexpressed in primary samples derived from COVID-19 infected patients and COVID-19 mouse models. Importantly, rare germline activating variants in the NEDD4 and WWP1 genes are associated with severe COVID-19 cases. Critically, I3C, a natural NEDD4 and WWP1 inhibitor from Brassicaceae, displays potent antiviral effects and inhibits viral egression. In conclusion, we identify the HECT family members of E3 ligases as likely novel biomarkers for COVID-19, as well as new potential targets of therapeutic strategy easily testable in clinical trials in view of the established well-tolerated nature of the Brassicaceae natural compounds.