RESUMEN
SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is the key enzyme required for viral replication and mRNA synthesis. RdRp is one of the most conserved viral proteins and a promising target for antiviral drugs and inhibitors. At the same time, analysis of public databases reveals multiple variants of SARS-CoV-2 genomes with substitutions in the catalytic RdRp subunit nsp12. Structural mapping of these mutations suggests that some of them may affect the interactions of nsp12 with its cofactors nsp7/nsp8 as well as with RNA substrates. We have obtained several mutations of these types and demonstrated that some of them decrease specific activity of RdRp in vitro, possibly by changing RdRp assembly and/or its interactions with RNA. Therefore, natural polymorphisms in RdRp may potentially affect viral replication. Furthermore, we have synthesized a series of polyphenol and diketoacid derivatives based on previously studied inhibitors of hepatitis C virus RdRp and found that several of them can inhibit SARS-CoV-2 RdRp. Tested mutations in RdRp do not have strong effects on the efficiency of inhibition. Further development of more efficient non-nucleoside inhibitors of SARS-CoV-2 RdRp should take into account the existence of multiple polymorphic variants of RdRp.
RESUMEN
RNA-dependent RNA polymerase (RdRp) plays a key role in the replication of RNA viruses, including SARS-CoV-2. Processive RNA synthesis by RdRp is crucial for successful genome replication and expression, especially in the case of very long coronaviral genomes. Here, we analysed the activity of SARS-CoV-2 RdRp (the nsp12-nsp7-nsp8 complex) on synthetic primer-templates of various structures, including substrates with mismatched primers or template RNA modifications. It has been shown that RdRp cannot efficiently extend RNA primers containing mismatches and has no intrinsic RNA cleavage activity to remove the primer 3'-end, thus necessitating the action of exoribonuclease for proofreading. Similar to DNA-dependent RNA polymerases, RdRp can perform processive pyrophosphorolysis of the nascent RNA product but this reaction is also blocked in the presence of mismatches. Furthermore, we have demonstrated that several natural post-transcriptional modifications in the RNA template, which do not prevent complementary interactions (N6-methyladenosine, 5-methylcytosine, inosine and pseudouridine), do not change RdRp processivity. At the same time, certain modifications of RNA bases and ribose residues strongly block RNA synthesis, either prior to nucleotide incorporation (3-methyluridine and 1-methylguanosine) or immediately after it (2'-O-methylation). The results demonstrate that the activity of SARS-CoV-2 RdRp can be strongly inhibited by common modifications of the RNA template suggesting a way to design novel antiviral compounds.
RESUMEN
The SARS-CoV-2 betacoronavirus pandemic has claimed more than 6.5 million lives and, despite the development and use of COVID-19 vaccines, remains a major global public health problem. The development of specific drugs for the treatment of this disease remains a very urgent task. In the context of a repurposing strategy, we previously screened a library of nucleoside analogs showing different types of biological activity against the SARS-CoV-2 virus. The screening revealed compounds capable of inhibiting the reproduction of SARS-CoV-2 with EC50 values in the range of 20-50 µM. Here we present the design and synthesis of various analogs of the leader compounds, the evaluation of their cytotoxicity and antiviral activity against SARS-CoV-2 in cell cultures, as well as experimental data on RNA-dependent RNA polymerase inhibition. Several compounds have been shown to prevent the interaction between the SARS-CoV-2 RNA-dependent RNA polymerase and the RNA substrate, likely inhibiting virus replication. Three of the synthesized compounds have also been shown to inhibit influenza virus. The structures of these compounds can be used for further optimization in order to develop an antiviral drug.