Discovery of small molecule derivatives as SARS-CoV-2 nsp13 inhibitors: a new strategy to develop broad-spectrum antiviral agents

It has been more than four years since the first report of SARS-CoV-2, the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Several efforts have been focused on vaccine development in an exceptionally rapid time frame, as well as the evaluation of a wide range of potential treatments in clinical trials, a few of which have also reached the market. However, these drugs are characterized by several limits (including low response to treatment in some patients, low effectiveness against the new variants, severe side effects, etc), thus underscoring the need to speed up the research. Indeed, the development of specific inhibitors is of paramount importance for: i) emergence of virus strains evading vaccine antibody response; ii) prospect of new coronaviruses (CoVs) outbreaks in the future. Among potential drug targets, the SARS-CoV-2 non-structural protein 13 (nsp13) is highly attractive thanks to its pivotal role in viral replication.1 Nsp13, member of the 1B helicase superfamily, utilizes the energy of nucleotide triphosphate hydrolysis to catalyze the unwinding of double-stranded DNA or RNA in a 5′ to 3′ direction.2 Nonetheless, nsp13 is the most conserved non-structural protein within the CoV family.3 This implies that small molecule nsp13 inhibitors may serve as pan-CoV inhibitors and that previously found compounds targeting nsp13 in other CoVs might be effective against COVID-19. Although the pivotal role of nsp13, there is a paucity of information about small molecules reported in literature as nsp13 inhibitors. Basing on literature data and thanks to our longstanding expertise in the design and synthesis of small molecules endowed with antiviral activities, we explored SARS-CoV-2 nsp13 as drug target using our in-house library of compounds, identifying a promising hit as micromolar nsp13 inhibitor.4 We synthesized a set of derivatives structurally related with the identified hit, obtaining new dual SARS-CoV-2 nsp13 ATPase and unwinding inhibitors, also capable of inhibiting viral replication. Mode-of-action studies revealed non-competitive kinetics of inhibition, suggesting an allosteric binding. Docking studies suggested a possible binding mode within an allosteric pocket of the nsp13 enzyme. Moreover, the broad-spectrum antiviral activity was evaluated, highlighting the potential of these compounds as pan-CoV inhibitors. The data coming from the biological assays will be shown and discussed.