Development and characterization of novel antivirals against emerging viruses

Coronaviruses have gained a great deal of attention after the emergence of three highly pathogenic viruses in the last two decades, SARS-CoV, MERS-CoV, and SARS-CoV-2. The current state of the SARS-CoV-2 pandemic is shedding light on the coevolution of coronaviruses with humans and on the putative origin of the other endemic coronaviruses; nevertheless, the frequency of epidemic and pandemic events, as well as the disease severity and transmissibility of coronaviruses, is expected to pose significant challenges in the near future. While vaccine development has made remarkable advances in the COVID-19 pandemic, antivirals for treating severe disease are still needed. The development of broad-spectrum anti-coronavirus antivirals acting with different mechanisms could be a starting point in the future to face pandemic events. In the context of developing novel antivirals, part of my thesis concerns the characterization of small-molecule antivirals acting on the SARS-CoV-2 Main protease (Mpro). A large body of literature has shown the effectiveness of targeting Mpro. Indeed, one of the few small-molecule inhibitors currently approved for the treatment of COVID-19 is the Mpro inhibitor Nirmatrelvir, a catalytic inhibitor of the enzyme. The projects I participated in were focused on the identification of alternative mechanisms to target this viral enzyme, in particular allosteric inhibition and degradation through proteolysis targeting chimeras (or PROTACs). Compounds were first screened for their activity against SARS-CoV-2 in Vero E6 cells by plaque reduction assays, and toxicity was determined through MTT assays. Compounds were also tested against two endemic human coronaviruses, HCoV-OC43 and HCoV-229E, as well as against different SARS-CoV-2 variants, to evaluate the presence of broad-spectrum activity. Then, the putative allosteric inhibitors were investigated for their ability to inhibit Mpro in vitro and in cellular contexts and were then tested for their ability to interfere with the Mpro monomer-dimer equilibrium. Concerning the PROTAC degraders, the most active compounds were tested for their ability to induce the degradation of Mpro in a cellular context, and the dependency on the Von Hippel-Lindau ubiquitin ligase was demonstrated. Furthermore, it was possible to demonstrate the biophysical binding of the hit compounds with the target protein Mpro through microscale thermophoresis analysis (MST). A second line of my research project concerned the characterization of inhibitors of the NS3-NS5 interaction from dengue virus (DENV). DENV is an emerging flavivirus considered a public health risk since climate change and increased human mobility have greatly expanded the geographical distribution of the vector mosquitoes of the Aedes genus. The NS3-NS5 interaction has been previously demonstrated to be essential for the formation of a functional polymerase complex, and inhibitors of this interaction are characterized by a broad-spectrum activity against DENV serotypes and, more generally, against flaviviruses. Indeed, previous work from other members of our research group successfully identified NS3-NS5 inhibitors active in plaque reduction assays against DENV-2 and able to disrupt the NS3-NS5 complex formation by an ELISA-based assay. During my PhD, I worked on the biophysical characterization of the interaction between NS3 and NS5 and demonstrated through MST analysis the interaction between the hit compound 3 and NS5, which corroborates the data obtained in vitro and in silico. Furthermore, I began to work on characterising the broad-spectrum activity of the compounds against flaviviruses, evaluating their activity against Zika virus.