Relevance of the viral Spike protein/cellular Estrogen Receptor-α interaction for endothelial-based coagulopathy induced by SARS-CoV-2

Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor- (ER). Given the implications of ER in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ER, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo, without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.

Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.

Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants

The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease, which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach. Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with mild to moderate COVID-19.