RNA DEPENDENT RNA POLYMERASE: A VALUABLE TARGET TO BLOCK VIRAL REPLICATION IN SINGLE-STRANDED (+)SENSE RNA VIRUSES.

The (+)strand RNA viruses include a very large group of viruses that cause epidemic diseases in humans, including dengue fever and gastroenteritis. The human (+)RNA viruses include Flaviviruses (FV) and Norovirus (NV). Both encode for proteins essential for viral replication, such as the RNA dependent RNA polymerase (RdRp). Since human cells lack RdRp, it appears as one of the most promising targets for antivirals development. I worked on the identification of new non-nucleotide inhibitors against FV and NV, using RdRp as the main target. In this context, suramin and NF023 have been identified in my lab as NV RdRp inhibitors that, however both are hampered in their application by pharmacokinetics problems. To overcome such problems, I analyzed the potential inhibitory role of Naf2, a fragment derived from these two molecules. Although Naf2 showed a low inhibitory activity, the crystal structures of NV RdRp/Naf2 complex revealed a new binding site. To further map this new site, I tested a Naf2 related molecule, PPNDS. The crystal structures of the RdRp/PPNDS complex revealed interesting features about the new binding site. I also focused on structurally related molecules synthesized following structure-driven information. NV RdRp crystal structures in complex with one of these compounds (Cpd6) were analyzed, providing new knowledge on the interactions between a small fragment and NV RdRps, establishing a platform for structure-guided drug optimization. In parallel to the NV work, I screened in silico a library of compounds against FV RdRp. One of the best compounds identified (HeE1-2Tyr) was able to inhibit the RdRp activity and several FVs in cell-based assays. Although the crystallographic analyses don't reveal clear enough electron density for the inhibitor, indirect evidence suggests that HeE1-2Tyr interferes with the RdRp priming loop that appears disordered.