Study of antiviral activity of diazadispiroalkane compounds against alpha- and beta- coronaviruses.

RNA viruses cause many human and animal infections characterized by worldwide spread, high mortality, and significant health costs. In the case of some RNA virus diseases, such as Polio, Measles, Yellow Fever, and others, there is the possibility of accessing effective immunoprophylaxis measures. Still, the incidence and severity of some viral diseases are to develop effective and selective therapies. This scenario is essential to develop and manufacture new and effective antiviral agents. Usually, existing antiviral agents target specific viral proteins, with a higher probability of developing drug-resistant, especially those with an RNA genome. The recent SARS-CoV-2 pandemic has moved the scientific world to pay particular attention to the Coronaviridae family, ssRNA (+) viruses that cause respiratory diseases. We focused our studies on Alphacoronavirus HCoV-229E and Betacoronavirus HCoV-OC43 and SARS-CoV-2. These viruses use different cell receptors to enter the host cell. Recent studies have highlighted an additional mechanism of endocytosis upstream of the binding with the receptor, which involves the heparan sulfate (HS) present on the cell surface through an electrostatic interaction in which negative HS charges interact with the virus envelope, concentrating the virions and allowing the recognition of the secondary receptor. Therefore, this phase of the entry process is strategically interesting as it could be the target of broad-spectrum antivirals. Among the strategies used to block the HS-virus interaction, one particularly effective is the use of small cationic molecules that bind competitively to HS. Among these cationic molecules of particular interest are the dispirotripiperazines and their diazadispiroalkane derivatives synthesized and kindly provided us by Prof. Vadim Makarov (Russian Academy of Sciences) and colleagues. During my Ph.D. course, the main goal was to identify new antiviral compounds with broad-spectrum activity and study their mechanism of action. These objectives have been achieved through a multidisciplinary approach. We evaluated the toxicity and the antiviral activity of some diazadispiroalkanes, against HCoV-229E, -OC43, and SARS-CoV-2. Subsequently, we then evaluated the selectivity and potency of these compounds, studied its mechanism of action, examined the kinetics of virus adsorption in the presence of the compound. After all, we verified whether our identified lead derivative could interfere with the penetration of the virus in the cells. The lead compound was shown to be a SARS-COV-2 inhibitor and more robust against HCoV-OC43. The molecule acts during the pre-treatment and in the early stages of infection, causing a dose- dependent reduction in infectivity. Therefore, the validated method for evaluating the antiviral activity of diazadispiroalkanes can be applied for the discovery and development of broad-spectrum antiviral drugs in vitro.