Coronaviruses have been responsible for several global disease outbreaks over the last 20 years, including the “Severe Acute Respiratory Syndrome” in 2002/2003, the “Middle East Respiratory Syndrome” in 2012, and the “Coronavirus Disease of 2019 (COVID19)”. These viruses are highly contagious and can cause multiple medical disorders upon contraction, such as common cold or lower respiratory infections. SARS-CoV-2, the newly emerged coronavirus variant of 2019, has been confirmed as the cause of the ongoing COVID19 pandemic, which infected over 167 million people worldwide and, by the end of May 2021, has a death toll of over 3 million people. Even though several SARS-CoV-2 vaccines have made it to the market, no proven options have yet been discovered for treating COVID19 infections. The aim of this project is, therefore, to improve the potency of two active SARS-CoV-2 main protease (Mpro) inhibitors (ML188 and X77) by performing a structure-activity-relationship study where two specific sites of the inhibitors are altered. The inhibition activity of these compounds is then tested on isolated SARS-CoV-2 Mpro. The four-component Ugi reaction was utilized to synthesize the ML188 and X77 analogs, which were purified by column chromatography before testing. During this project, six pure analogs were successfully synthesized and will be sent shortly for testing. Inhibitors with good activity against SARS-CoV-2 Mpro will be further tested for their antiviral activity in cell-based infection assays. The results obtained from this study will later be used to perform a second structure-activity-relationship study to further improve the potency of the two inhibitors by developing a 2nd generation library.

Coronaviruses have caused three large outbreaks in the past century. The most recent one, also still ongoing, is represented by the SARS-CoV-2/Covid-19 pandemic. Efforts have been taken to develop efficient vaccines and antivirals and one of the major virus-based targets in drug development is represented by the main protease of these viruses. Main proteases are proteins (cysteine hydrolases) with high level of conservation among different coronaviruses and have an important role in the virus life cycle. Due to the need of developing broad-spectrum antivirals against Coronaviruses, this study aimed to set up a CPE-based assay for testing compounds against the main protease of human coronavirus 229E. An optimized TCID50 protocol was established by using MRC-5 cells, at a density of 1x104 cells/ml with a 3h incubation prior infection with a concentration of 10-1 of HCoV-229E. The cell viability was assessed through MTT assay. Using reference compounds, with previously demonstrated antiviral potency against the main protease of different coronaviruses (GC-376, Nirmatrelvir), the efficiency of the conceived assay was validated (GC-376 EC50 = 1.24 μM; Nirmatrelvir Ec50= 0.72 μM). Compound 19 was proved to also be active against the main protease of HCoV-229E (EC50 = 0.22 μM), and together with previous findings, it was concluded that this compound has a broad-spectrum activity. Newly developed compounds MP17 and MP19 were also demonstrated to be efficient against HCoV-229E. As a future perspective, further investigations of these compounds should take place for the identification of EC50 values.

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