Unveiling the antiviral capabilities of targeting Human Dihydroorotate Dehydrogenase against SARS-CoV-2 (preprint)

The urgent need for effective treatments against emerging viral diseases, driven by drug-resistant strains and new viral variants, remains critical. We focus on inhibiting the human dihydroorotate dehydrogenase (HsDHODH), one of the enzymes in charge of pyrimidine nucleotide synthesis. This strategy could impede viral replication without provoking resistance. We evaluated quinone-based compounds, discovering potent HsDHODH inhibition (low nanomolar IC50) and promising in vitro anti-SARS-CoV-2 activity (low micromolar EC50). These compounds exhibited low toxicity, indicating potential for further development. Additionally, we employed computational tools like molecular docking and QSAR models to analyse protein-ligand interactions. These findings represent a significant step forward in the search for effective antiviral treatments and have great potential to impact the development of new broad-spectrum antiviral drugs.

Identification and preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral and anti-inflammatory therapy (preprint)

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still scarce and the emergence of new variants challenging immunized individuals, suggests that mutant viruses might also emerge because of antiviral pressure. Therefore, beyond the recently alleged positive antiviral clinical results with molnupiravir™ and paxlovid™, the continuous search for drugs against 2019 coronavirus disease (COVID-19) is necessary. Because severe COVID-19 is a virus-triggered immune and inflammatory disfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that N6-furfurylaminopurine (kinetin, MB-905) inhibits the in vitro replication of SARS-CoV-2 at the sub-micromolar range in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. As a pro-drug, MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce an error-prone virus replication. Consistently, co-inhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously placed nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. SARS-CoV-2-infected transgenic mice expressing human ACE2 were treated with MB-905 and decreased viral replication of the gamma variant was observed, along with reduced lung necrosis, hemorrhage and inflammation, together with increasedmice survival. MB-905 showed good oral absorption, its metabolites were stable and achieved long-lasting plasma concentrations exceeding those required for the in vitro inhibition. Besides, MB-905 was neither mutagenic, toxic during chronic treatment, nor cardiotoxic. Because kinetin has already been clinically investigated for a rare genetic disease at regimens that are beyond the predicted concentrations of antiviral/anti-inflammatory inhibition demonstrated here, our investigation strongly suggests the opportunity for a rapid clinical development of a new and orally available antiviral substance for the treatment of COVID-19.

Atazanavir is a competitive inhibitor of SARS-CoV-2 Mpro, impairing variants replication in vitro and in vivo (preprint)

Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19), however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based, and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (M pro ) was inhibited by ATV, with Morrison’s inhibitory constant (Ki) 1.5-fold higher than boceprevir (GC376, a positive control). ATV was a competitive inhibition, increasing the M pro ’s Michaelis-Menten (K m ) more than 6-fold. Cell-based assays indicated that SARS-CoV-2 gamma is more susceptible to ATV than its predecessor strain B.1. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the M pro /ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.

A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories (preprint)

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

Human endogenous retrovirus K activation in the lower respiratory tract of severe COVID-19 patients associates with early mortality (preprint)

Critically ill 2019 coronavirus disease patients (COVID-19) under invasive mechanical ventilation (IMV) are 10- to 40-times more likely to die than the general population. Although progression from mild to severe COVID-19 has been associated with hypoxia, uncontrolled inflammation and coagulopathy, the mechanisms involved in progression to severity are poorly understood. By analyzing the virome from tracheal aspirates (TA) of 25 COVID-19 patients under IMV, we found higher levels and differential expression of human endogenous retrovirus K (HERV-K) genes compared to nasopharyngeal swabs from mild cases and TA from non-COVID patients. Proteomic analysis and RT-PCR confirmed the presence of HERV-K in these patients. Moreover, increased HERV-K expression was triggered in human primary monocytes from healthy donors after experimental SARS-CoV-2 infection in vitro. In critically ill patients, higher HERV-K levels were associated with early mortality (within 14 days) in the intensive care unit. Increased HERV-K expression in deceased patients associated with IL-17-related inflammation, monocyte activation and higher consumption of clotting/fibrinolysis factors. Our data implicate the levels of HERV-K transcripts in the outcome of critical COVID-19 patients under invasive mechanical ventilation.