Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Commercially Available Flavonols Are Better SARS-CoV-2 Inhibitors than Isoflavone and Flavones.

Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3'-5' exonuclease (ExoN).

The Endolysosomal System: The Acid Test for SARS-CoV-2.

This review aims to describe and discuss the different functions of the endolysosomal system, from homeostasis to its vital role during viral infections. We will initially describe endolysosomal system's main functions, presenting recent data on how its compartments are essential for host defense to explore later how SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) and other coronaviruses subvert these organelles for their benefit. It is clear that to succeed, pathogens' evolution favored the establishment of ways to avoid, escape, or manipulate lysosomal function. The unavoidable coexistence with such an unfriendly milieu imposed on viruses the establishment of a vast array of strategies to make the most out of the invaded cell's machinery to produce new viruses and maneuvers to escape the host's defense system.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

Unlike Chloroquine, Mefloquine Inhibits SARS-CoV-2 Infection in Physiologically Relevant Cells.

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC50 of 1.2 µM and EC90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.