Development of new broad spectrum antiviral drugs against the nucleoprotein from Influenza A – FLUNUCLEOVIR

Structural virology: electronic microscopy, biophysical methods in solution, 3D crystallography of free and RNA-bound NP.
- Drug design: in silico screening of new inhibitors of NP (leads) and their iterative improvement
- Antiviral testing: in vitro, in cells & in vivo (rodent model)

- Expression, purification of monomeric wt NP and its monomeric or trimeric mutants
- X-ray crystallography of RNA-free NP
- Identification and testing of several inhibitors of NP

- Understanding of the interactions between NP and viral RNA, which is an essential part of the viral machinery required for Influenza A replication
- 3D structure of a complex NP-inhibitor allowing effective improvement of the inhibitor.

French patent: COMPOSITIONS ANTIVIRALES DIRIGEES CONTRE LA NUCLEOPROTEINE DES VIRUS INFLUENZA » Anny Slama-Schwok et Bernard Delmas

- B. Tarus, O Bakowiez, S.Chenavas, L.Duchemin, L. Estrozi, C. Chevalier, C. Bourdieu, J. Bernard, M.Moudjou, B. Delmas, C. Di Primo, R.WH Ruigrok and A. Slama-Schwok : Multiple oligomerization paths of the nucleoprotein from Influenza A virus Biochimie: 94 776-785. doi.10.1016/j.biochi.2011.11.009
- B. Tarus, C. Chevalier, B. Delmas, C. Di Primo, and A.Slama-Schwok. Molecular dynamics studies of the nucleoprotein from Influenza A virus , PlosOne 2012 (1): e30038. doi:10.1371/journal.pone.0030038
- Ruigrok RW, Crépin T, Kolakofsky D. Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Microbiol. 2011 Aug;14(4):504-10. Epub 2011 Aug 6.

The present pandemic spread of H1N1 Influenza A virus and the threat of the virulent H5N1 strains urgently require the development of new antivirals. The existing antiviral drugs target neuraminidase or the proton channel M2, both proteins with a relatively high mutation rate. Antivirals against the polymerase complex as ribavirin present a low specificity for Influenza A virus and often interfere with host cellular functions. New strategies for antiviral development target the polymerase complex and associated cofactors (Beigel, 2008). The nucleoprotein NP that covers the 8 viral genomic RNA segments has a key role in regulating transcription and replication of Influenza A virus. Because of the crucial role of Influenza nucleoprotein and its high conservation in all strains of Influenza A, NP is a promising target for antivirals that was never exploited to the best of our knowledge.
Aims: The main objective of this study is to develop novel inhibitors against the Influenza A viruses, in particular one that interferes with the binding of nucleoprotein to viral RNA. Here, we start with two new inhibitors of the NP protein already selected by in silico screening, based on the known X-ray structures of NP obtained in the absence of RNA (Ye et al., 2006; Ng et al., 2008). We have already shown that the newly identified compounds dock in the putative RNA binding groove of NP, compete with RNA binding; consequently, these compounds should inhibit RNA-dependant transcription and replication of Influenza A viruses. The identity of these compounds, named N and T, will remained hidden in this abstract
Our strategy will be -1 to solve the structures of NP-N, NP-T and NP-RNA complexes. The structure of nucleoprotein in complex with such an inhibitor will guide the chemical modifications of the inhibitor. -2 This will lead at designing a second generation of inhibitors with higher affinity for Influenza A NP and sufficient specificity for minimal interference with the host.
-1 Structural characterisation of the nucleoprotein of Influenza A and of the related Thogoto virus. The structures will be solved by the group of Rob Ruigrok at the UVHCI laboratory at Grenoble. The new structures of NP-RNA complexes of Influenza A and Thogoto will be compared with the previously reported complexes of rabies (Albertini et al. 2006) and respiratory syncitial viruses (Tawar et al. 2009), leading to (i) the control of NP oligomerization –(ii) more insight of RNP structure-function and (iii) in the conservation of nucleoproteins in the Orthomyxoviridae. -2 Novel, structure-based antiviral strategies targeting the nucleoprotein: By molecular modelling, side chains will be added to naproxen (fragment based design) and the stability of the complexes will be tested by molecular dynamics in explicit solvent by access to the IBM Vargas cluster of Orsay. The antiviral effects of the new inhibitors against various viral strains will be tested in vitro, in cell cultures and animal model in the P2 and P3 confinement labs of the unit of Virologie et Immunologie Moléculaires of l’INRA.
This project combines the complementary expertises in structural virology, molecular biology and protein biochemistry, biophysics and drug design and benefits from expertise in synthetic chemistry. New antiviral compounds with minimal interference of the host functions of transcription and replication should protect human and animal health against viral pathogens and have a high potential of exploitation.