Viruses of the Flaviviridae family are widespread vector-borne pathogens causing large epidemics. Members of the Flaviviridae family consist of a large group of enveloped viruses with a +RNA genome. Dengue virus (DENV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV) are all emerging or reemerging pathogens.
Their NS5 protein consists of RNA-dependent RNA polymerase (RdRp) subunit and methyltransferase (MTase) subunit that is responsible for N-7 and 2′-O methylation of the viral RNA cap which protects the RNA from being recognized by host sensors. We have already shown that the RdRp can be targeted by nucleotide analogs [1] and we performed structural analysis of the Zika MTase [2]. Now, we explore the possibility to target different (ZIKV, TBEV, DENV and YFV) MTase domains by the same compound.
We produced recombinant MTase domains in E. coli and assayed them enzymatically. A key step was the removal of S-adenosyl-L-methionine (SAM) - the methyl donor that co-purifies with the MTases. We have already designed several analogs of SAM and using thermalshift assay determined which of them bind into SAM-binding pocket. The best compound was crystallized with DENV Mtase and the crystal structure helped us with the design of new series of ligands.
In order to characterize dissociation constant of our compounds we developed a fluorescence anisotropy assay. For this purpose, we first had to prepare a fluorescently labeled analog of SAM and determine its affinity to the Mtase domain. Now ligand displacement assay (fluorescent ligand is displaced by a non-fluorescent ligand and the anisotropy of fluorescence goes down) can be used to measure affinity of any ligand towards the MTase [3].
The work was was supported by Czech Science Foundation grant number 17-05200S and also from European Regional Development Fund; OP RDE; Project: "Chemical biology for drugging undruggable targets (ChemBioDrug)" (No. CZ.02.1.01/0.0/0.0/16_019/.