CNRS - AIX MARSEILLE UNIV: UMR7257

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Lescar’s Team Projects

Fragment-based drug discovery using the NS3 and NS5 flavivirus domains as targets

People Involved : Dr Karine Barral, Fatiha Benmansour

Flaviviridae comprise major human pathogens that are grouped in three families: flaviviruses (Dengue virus (DENV), Yellow-fever virus, Japanese encephalitis virus) hepaciviruses (Hepatitis C virus) and pestiviruses. While vaccines are available that confer protection against some flaviviruses (eg: Yellow-fever and Japanese encephalitis), no vaccine has currently been fully validated conferring sufficient protection against the four strains of DENV that circulate in tropical and subtropical regions of the globe, putting several billions people at risk of contracting this potentially life-threatening disease with Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) being the severe forms affecting more than 200,000 people each year and provoking more than 20,000 deaths.

Our research program during the last years has consisted in studying several non-structural proteins (NS) from dengue virus using biochemical and structural biology methods with a view to provide a better understanding of their role in the virus life cycle (fig. 1). Thus, we could provide structures at atomic resolutions of the NS3 helicase domain, of the NS3 protease-helicase, of the polymerase domain of the NS5 protein as well as of the NS5 methyltransferase domain in complex with RNA and inhibitors. Of note, these enzymes constitute crucial targets for the identification and the development of compounds with antiviral activities, a task that we have started as a collaborative effort with the Novartis Institute for Tropical Diseases in Singapore, as well as with the team of Bruno Canard in Marseille.

Figure 1. Flavivirus genome organization
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Figure 2
Fragment-Based Drug Design strategy

Our objective is here to develop a Fragment Based Drug Design « FBDD » strategy to identify and optimize new inhibitors of Dengue virus.

Fragment-based drug design is a new approach that has been successfully applied to challenging targets. While 3-D protein structures have been used in drug discovery for many years now, fragment-based drug design uses x-ray crystallography or other biophysical techniques to screen fragment libraries for specific binding to a target protein (fig. 2). Knowledge of exactly how the fragments bind to the protein target allows the hits to be extended (or linked with other fragments) to create higher affinity compounds by round of improvements using medicinal chemistry and structure-based drug design (computational and combinatorial chemistry experiments).

In our lab, we use Biophysical methods (ITC, SPR, Thermofluor®) as first-line fragments libraries screening to identify small fragments (typically of mass less than 300 Da) that bind to the NS3 or NS5 dengue virus domain. We then co-crystallize NS3 or NS5 dengue virus crystals with selected fragments to solve the high-resolution structures of complexes. This work is followed by medicinal chemistry approaches whereby the fragment is extended based on the interactions observed in the crystal structure of the complex (Fig. 3). This approach should lead to antiviral compounds with a higher binding affinity to the target protein. Enzymatic assays are performed in parallel to assess the inhibitory activity of these new inhibitors.

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Figure 3
Frangment optimization

Collaborators

- Dr Gérard Bricogne (Global Phasing, Cambridge, UK)
- Dr Andrea Brancale (University of Cardiff)
- Dr Bruno Coutard (AFMB)
- Dr Etienne Decroly (AFMB)
- Dr Bruno Canard (AFMB)
- Dr Gilles Querat (IRD-UMR190)

Funding EU-FP7 SILVER European consortium

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project supported by the French National Agency for Research (ANR-13-JS07-0006-01)

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