Discovery of three small-molecule inhibitors targeting Ebolavirusgenome replication and transcription

Using established ligand discovery pipeline, three small molecule inhibitors were identified to target hydrophobic binding pocket of the Ebola virus nucleoprotein. These are MCCB5, CB6  and MJM364. The compounds interfere with the NP VP35 protein protein interaction required for viral replication and transcription. Further evaluation using EboV minigenome assay showed a potent dose dependent inhibition with EC50 values of 4.41 µM, 0.194 µM and 0.834 µM for MCCB5, CB6 and MJM364 respectively in BSR-T7 cells. These concentrations also showed low cytotoxicity The drugs were confirmed to be specific for EBOV as they showed a lack of efficacy against Bunyamwera virus. The inhibitory activity was also shown  and validated in human hepatoma cells, Huh7-Lunet-T7. Time of addition studies showed that MCCB5 suppressed minigenome activity when administered posttransfection about 24-48 hpt showing its capacity to target already established NP VP35 replication complexes which is a key therapeutic requirement for post exposure treatment. Mutation targeting hotspot 2 of the NP hydrophobic pocket caused  a shift to higher EC 50 values for all the three compounds showing that this pocket is their functional target. Since these NP residues are highly confirmed in filoviruses, these compounds are potential broad spectrum filoviral therapeutics. 
(MCW)