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Hit-to-Lead optimisation. Target : Dengue NS5 polymerase

Head Karine ALVAREZ

People involved : Dr Karine Alvarez, Dr Karine Barral, Fatiha Benmansour
PCML Platform : Dr Cécilia Eydoux, Prof JC Guillemot, Dr Nicolas Masse, Dr Barbara Selisko, Dr Laétitia Sthul-Gourmand

We performed a screening of 17,000 compounds on the NS5 polymerase of dengue virus. It ran as follows:
- The primary screening of all compounds (concentration of 35 to 100 µM)
- The secondary screening of compounds selected in the first round (threshold to 78% inhibition)
- Confirmation of the activity of the compounds on batchs of fresh powder.
- The characterization of the compounds (NMR, MS, HPLC-MS coupling)
- The determination of IC50 on the target.
- Evaluation of the activity of the compound on dengue replicon assay. The molecules identified, confirmed and characterized are tested to assess their replicon activity and cytotoxicity in cells.
- research of families of compounds and evaluation of the interests of development.
- Selection of the most interesting hits for extensive biochemical characterization and synthesis of analogs (hit-to-lead optimisation).

From 149 hits, 96 compounds were obtained in fresh powder, 55 compounds were confirmed as active, 44 compounds were characterized, their structure and purity were determined, 30 compounds have shown a good IC50 and we were interested in 4 of them for which we try to characterize the determinants of antiviral activity. To optimize the pharmacological profile of these "hits" and improve their activity, specificity, solubility, toxicity, we initiated a project "hit-to-lead optimization" internally and in collaboration with some collaborators from the National Library (Pr Marcel Hibert, Dr.F.Gueritte, Dr.F.Mahuteau, Dr.S.Piguel).

In the team, we focused our efforts to optimize a target molecule from the chemical library of the Institut Curie. We first, pushed the characterization of the hit in terms of specificity towards other viral polymerases, mode of action and activity in replicon assay. On this basis, our goal was to improve the pharmacological properties and decrease the toxicity of the molecule particularly high in replicon assay, while enhancing its activity against the enzyme.

Two hundred molecules were synthesized, 144 were tested. The most promising compounds were tested in replicon assay and those with no toxicity were sent for testing in infected cell cultures with multiple serotypes of dengue (DEN-1 to DEN-4) (collaboration Dr. Gilles Querat, SILVER project). To date, we have supported the improvement of a hit to a marked decrease in cell toxicity, while retaining good activity of the enzyme. We hope so, to develop expertise and methods that can be applied to other molecules and to other viral targets. We also wish to increase our “in house” library. Ultimately, we hope to set up a platform of synthesis integrated in a multidisciplinary setting dedicated to antiviral drugs. We have equipment for parallel synthesis and purification, a microwave synthesizer, and a system HPLC / MS for the analysis of the synthesized compounds.

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Figure 1
Parallel synthesis apparatus BUCHI
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Figure 2
Parallel Purification BIOTAGE
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Figure 3
Micro-wave synthetizeur BIOTAGE
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Figure 4
Analysis HPLC/MS Thermo-Scientific


- Masse N, Davidson A, Ferron F, Alvarez K, Jacobs M, Romette JL, Canard B, Guillemot JC (2010) Antiviral Res 86 296-305


- Dr Françoise Gueritte, ICSN, Gif-sur-Yvette, France.
- Dr Florence Mahuteau et Dr Sandrine Piguel, Institut Curie Recherche, Orsay, France.
- Pr Marcel Hibert, Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081, Illkirch Cedex, France
- Dr Xavier Morelli, Laboratoire de Bioénergéntique et Ingénierie des protéines (UPR9036), Marseille, France.
- Dr Gilles Querat, Unité des virus émergents, Faculté de Médecine, Marseille, France.


- Projet Européen SILVER. FP7-HEALTH-2010-Drug lead discovery against RNA viruses. Section 3. Contrat CDD Ingénieur d’Etude en chimie de 12 mois dans le cadre du contrat Européen SILVER (Février 2012-).
- Projet ANR-07-BLAN-0285. Dengue D.-D. (2007-2010). 200000 euros sur 3 ans.

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