The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

30SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost 31 importance to understand the virus biology and to rapidly assess the potential of existing drugs 32 and develop new active compounds. While some animal models for such studies are under 33 development, most of the research is carried out in the Vero E6 cells. Here, we propose fully 34 differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. 35 Further, we also provide basic characteristics of the system. 36 37While these viruses are present in the human population for a long time, they are believed to 43 enter the human population in a zoonotic event, and one may speculate that they may have 44 caused epidemics similar to the one observed for the SARS-CoV-2. Time to the most recent 45 ancestor analysis suggests that human coronavirus HCoV-NL63 is the oldest species in humans, 46 followed by its cousin HCoV-229E and two betacoronaviruses, which emerged in humans in a 47 relatively near past 1,2, 3,4 . In the 21 st century, we already faced the emergence of the three novel 48 coronaviruses in humans, of which SARS-CoV disappeared after one season never to come 49 back, and MERS-CoV never fully crossed the species border, as its transmission between 50 humans is highly ineffective 5,6,7 . The 2019 zoonotic transmission, however, resulted in the 51 emergence of a novel human coronavirus, which seems to carry an optimal set of features 52 allowing for its rapid spread with considerable mortality. Whether the virus will become 53 endemic in humans is an open question 8,9,10 . 54At present, the studies on the virus are carried out using a surrogate system based on the 55 immortalized simian Vero E6 cell line 11 . While this model is convenient for diagnostics and 56 testing of some antiviral drugs, it has serious limitations and does not allow for the 57 understanding of virus biology and evolution. To make an example, the entry route of human 58 coronaviruses varies between the cell lines and differentiated tissue, not mentioning the immune 59 responses or virus-host interactions 12,13,14 . 60Here we used the fully differentiated epithelium cultures to study the infection with the 61 novel human coronavirus SARS-CoV-2. We observed an efficient replication of the virus in 62 the tissue, with the maximal replication at 2 days post-infection. At the time of the study no 63 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.antibodies were available. Therefore we developed immuno-FISH to show that the virus infects 64 primarily ciliated cells of the respiratory epithelium. 66Results and discussion 67 The HAE cultures reconstitute the tissue lining the conductive airways of humans. Fully 68 differentiated, are among the best tools for studying the viral infection in a natural 69 microenvironment 15 . These air-liquid interphase cultures contain a number of cell types (e.g., 70 basal, ciliated, and goblet). At the sa...

Background: This study presents the synthesis and multi-target behavior of the new 5'-hydroxy-3-(chalcogenyl-triazoyl)-thymidine and the biological evaluation of these compounds as antioxidant and anti-HIV agents. Objective: Antiretroviral therapy induces oxidative stress. Based on this, the main objective of this manuscript is the preparation of compounds that combine anti-HIV and antioxidant activities. Methods: The compounds were prepared from commercially available AZT, through a copper-catalyzed Huisgen 1,3-dipolar cycloadditions exploiting the AZT azide group and chalcogenyl alkynes. Results: The chalcogenium-AZT derivatives were obtained in good yields via click chemistry. The compounds evaluated showed antioxidant and anti-HIV activity. Additionally, in vivo toxicity of this class of compounds was also evaluated and the representative nucleoside did not change the survival, behavior, biochemical hepatic, and renal markers compared to the control mice. Conclusion: Data suggest the feasibility of modifying the AZT nucleus with simple organochalcogen fragments, exploring the reactivity of the azide group via 1,3-dipolar Huisgen cycloaddition reaction. The design of these new compounds showed the initially desired biological activities.

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