Identification and Characterization of the Mechanisms of Action of Novel Antiviral Compounds Targeting the Minimal Replication-Transcription Complex of SARS-CoV-2

After its emergence in 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent of COVID-19, a potentially fatal respiratory illness in Wuhan, China, which rapidly spread to the entire world. The pandemic has had dramatic economic, social and health-related consequences, with over 7 million deaths worldwide. SARS-CoV-2 belongs to the β-coronavirus genus in the Coronaviridae family, characterized by an enveloped nucleocapsid that contains one of the largest known RNA genomes with its ~30 kb in length. Among the viral proteins exploited for drug development, the RNA polymerase RNA dependent (RdRp) is the pharmacological target of two of the three drugs currently in clinical use for the treatment of COVID-19, namely remdesivir and molnupiravir. However, the absence of strong evidence of in vivo efficacy and safety still calls for the development of novel safe and potent antiviral drugs, that could combat this and other future threats from emerging coronaviruses. In the present work, we purified the viral proteins and set up a biochemical assay for the identification and molecular characterization of different classes of SARS-CoV-2 RdRp inhibitors. In collaboration with Dompé Farmaceutici S.p.A., we identified 42 novel non-nucleos(t)ide inhibitors of SARS-CoV-2 RdRp by in silico screening and selection of the best hits from a large library of >250.000 molecules, which contained both natural and repurposed drugs. We then assessed the mechanism of action of the two most potent ones, which showed nanomolar/low micromolar potency against the enzymatic RdRp activity. We showed how the prediction of the different binding sites of the two molecules is supported by experimental findings of their inhibitory mode in the biochemical assay. Further evaluations of the compounds against SARS-CoV-2 replication in a cell-based assay showed promising activity for their development as antiviral drugs. In the following chapter of results, in collaboration with prof. Chris Meier (University of Hamburg), a set of triphosphate nucleotide analogues, modified with the addition of a monoalkyl chain in the γ-phosphate, highlighted a novel mechanism of action. These analogues, in fact, were not incorporated by the polymerase into the nascent strand and did not compete with either RNA or NTP substrates. Further investigation into their mechanism of action suggested that these analogues may act with an allosteric binding mode to disrupt the minimal replication and transcription complex (RTC) of SARS-CoV-2. In addition, the generation of prodrug derivatives showed promising activity against SARS-CoV-2 replication in vitro. Lastly, in collaboration with prof. Fabrizio Manetti (University of Siena) we evaluated the effects of structural modifications of a series of suramin derivatives against the RdRp activity and viral replication of SARS-CoV-2 in cell culture. By assessing the effects of different sequential structural modifications introduced to the scaffold of the derivatives, we identified novel compounds with nanomolar potency against the enzymatic function and low micromolar activity against viral replication in vitro, providing the structural basis for the development of promising more potent and safe antiviral alternatives.