Identification of novel pyrimidine-based compounds as SARS-CoV-2 RNA-dependent RNA polymerase inhibitors.

For the fi6h consecu8ve year since 2019, the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infec8ons con8nues, leading to an increasing number of coronavirus disease 2019 (COVID-19) cases worldwide. The primary focus for scien8sts worldwide is to find at list one potent an8viral agents capable of effec8vely hal8ng or disrup8ng SARS-CoV-2 transmission, cellular entry, progressive replica8on, and pathogenicity, in the ongoing baOle against the resilient COVID-19 disease. However, the pool of agents primarily addressing the an8-replica8on requirements remains limited. Notably, compounds featuring nitrogen-based heterocyclic aroma8c cores in their structures, such as nucleoside-like compounds (nucleoside analogs), oxadiazoles, thiadiazoles, triazoles, quinolines, isoquinolines, and certain polyphenolics, have demonstrated significant promise as SARS-CoV-2 inhibitors. 1 Among these, the FDA-approved medica8ons molnupiravir and nirmatrelvir stand out. RNA-dependent RNA-polymerase, which has no counterpart in human cells, is an excellent target for drug development. 2 Small molecules targe8ng this protein are suitable for different mo8ves: - RdRps inhibitors holds the poten8al of ac8vity against various viruses because the target is conserved and unlike protease inhibitors, exhibit broader ac8vity because not all viruses encode a protease; - Polymerases present a high natural gene8c barrier to drug resistance due to their conserved nature, making polymerase inhibitors a promising op8on for developing broad-spectrum an8viral agents; - The current therapy against RdRp lacks small molecules because most of the inhibitors are nucleos(t)ide analogues administrated by iv (e.g. Remdesivir) or orally even if they showed several side effect (e.g. Molnupiravir). For this reason, we decided to invest our effort for the iden8fica8on of novel nitrogen-based small molecules targe8ng RdRp. In this contest, we decided to perform a large screening of the an8viral in-house compounds previously synthe8zed 3-4 . We focused our aOen8on over non nucleos(t)idic compounds with a pyrimidine central core that is well known as scaffold used for inhibitors of viral polymerases 5 . From this preliminary screening, several compounds were tested on RdRp SARS-CoV-2 Primer-elonga8on assay on PAGE and on those who showed a residual enzyma8c ac8vity (up to 30 μM) below 50% a6er the treatment, SARS-CoV-2 RdRp IC50 ± SD (μM) was determinate. Surprisingly, some compoundsshowed interes8ng in vitro ac8vity and similar IC50 with the drug used as control Simeprevir. A class of compounds characterized by a pyrimidine central core, showed the best ac8vity against RdRp. Our best compound from this series, RDS 4020, showed an IC50 of 5.88 ± 0.56 μM and was considered our HIT compound. To fully elucidate the mechanism of ac8on of our compound, co-crystal of RDS 4020 with the replisome of SARs-COV-2 by CryoEM experiment is ongoing. In the mean8me, we designed and synthe8zed novel deriva8ves of RDS 4020 that are currently under biological evalua8on to understand more about structure-ac8vity rela8onships (SARs) and to obtain a novel series of pyrimidine to be tested against other viral RdRps. The data coming from the screening will be shown and discussed.