Pan Nuclease Viral Inhibitors – PaNuVi

Bunyavirales and Arenaviridae are segmented negative strand single-stranded RNA viruses (sNSV) including many serious neglected and/or emerging human pathogens (e.g. respectively Lassa Fever (LASV), Junin (JUNV), Rift valley Fever (RVF), Crimean-Congo Hemorrhagic Fever (CCHF) and Hantaan viruses). The current lack of vaccine and limited therapeutic options for them make the development of efficacious drugs of high priority. The importance of providing effective therapeutic tools for emergent virus has been recently highlighted by the outbrakes of Ebola and Zika in the past few years.
The project will address this need for antiviral treatment with a new strategy based on the inhibition of endonuclease activity (EndoN) that these viruses carry essential for their replication cycle and in parallel with the exonuclease activity (ExoN) that carry only arenaviruses to escape the immune system. These two activities (EndoN and ExoN) are very specific mechanism, absent in the cellular biochemistry, and therefore formidable targets for antivirals by reducing the eventual cross-interaction with cellular enzymes.
Recently, several EndoNs domains from arenavirus (LASV, LCMV) and bunyavirus (LACV, Toscana, Hantaan), as well ExoN of Mopiea virus, have been structurally and functionally characterized by the partners1,2,3 of this proposal, showing that EndoNs have a similar architecture and that hydrolysis of RNA substrate by EndoN and ExoN follows a similar mechanistic involving two metal ions in a catalytic triade.
Our program aims to deliver a “pan-genus antiviral library” against human pathogenic arena- and bunyaviruses.
For this purpose, we propose a multidisciplinary approach settling a comprehensive pipeline of compound synthesis by applying a battery of state-of the-art chemistry, biochemistry, structural biology and virology to identify and improve compounds. We have developed an in-vitro fluorescent assay compatible with a HTS assay functional for EndoN activity. Compounds will undergo successive rounds of activity and affinity characterization followed by structure-guided optimization. The most promising candidates will then be tested on viruses in infected cells cultures to assess their inhibitory power and toxicity. This approach from in vitro screening to in cellula viral infection will allow us to rationally improve compounds and identify candidates with the required drug-like properties.
The project is heavily buttressed by the expertise and complementary of the partners (chemistry, biochemistry, crystallography and cellular virology), and promising preliminary results. Indeed, by structural and functional studies we have already identified a class of compounds targeting the metal ions for specifically binding the catalytic sites of EndoNs and ExoN4. Preliminary results show binding, target inhibition and activity in infected cells with an acceptable toxicity profile. This is already an optimal basis for the next steps of developing more specific and active compounds. The extremely wide expertise coverage of the project will boost the chances of success and speed-up the development process.

1. M. Saez-Ayala, et al. Crystal structures of LCMV endonuclease domain complexed with diketo acid ligands. IUCrJ, 2018, DOI: doi.org/10.1107/S2052252518001021, In Press.
2. J. Reguera, et al. Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching endonucleases. PLoS Pathog, 2016, 12, e1005636.
3. E. Yekwa, et al. Activity inhibition and crystal polymorphism induced by active-site metal swapping. Acta Crystallogr D 2017, D73, 641-649.
4. M. Saez-Ayala, E. Laban Yekwa, C. Mondielli, L. Roux, F. Bailly, P. Cotelle, D. Rogolino, B. Canard, S. Emonet, F. Ferron and K. Alvarez. Metal chelators for the inhibtion of the lymphocytic choriomeningitis virus endonuclease domain. Antiviral Res, 2018, in review.