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Dinucleotides as potent inhibitors of the HCV NS5B Polymerase

Head Karine ALVAREZ

People involved : Dr Karine Alvarez, Reuben Ovadia, Dr Stéphane Priet, Joelle Boretto-Soler

The objective of this project is to develop new treatments more effective against chronic hepatitis C to stop the disease progression to cirrhosis and liver cancer. We want to develop novel inhibitors type “nucleotide analogues” targeting replicative complex of hepatitis C and in particular the NS5B polymerase. Separation of RNA synthesis in two steps biochemically and structurally distinct opens the possibility to design inhibitors able to target one and/or both phases of initiation and elongation. We chose to design inhibitors that specifically target the initiation phase. We designed several series of "dinucleotides" GpC: a series of "dinucleotide phosphoramidate" and a series of "PNA chimera". (See Figure 1)

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Figure 1
Structures of target molecules : dinucleotide phosphoramidates, PNA and PNA chimera

The synthesis of phosphoramidates GpC dinucleotides, was conducted by Dr. Ivan Zlatev during his thesis under the supervision of Dr. Francois Morvan in the team of Dr. Jean-Jacques Vasseur (Laboratory of Biomolecular Chemistry Synthesis in Montpellier). Initially, 14 GpC dinucleotide phosphoramidates "3’-OH ’were synthesized, tested on the enzyme and HCV replicon assay. The most active compound obtained in this series shows an IC50 against the enzyme of 25 µM and an EC50 of 9 µM in replicon assay. (See Figure 2). Molecular modeling results obtained by Ivan Barvik show that the phosphoramidate dimer blocks the entrance of the nucleoside triphosphate to the active site leading to inhibition of the polymerization.

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Figure 2
Molecular model of dinucleotide phosphoramidate in the active site of HCV NS5B polymerase

In order to further increase the activity of these phosphoramidate dinucleotides GpC, a second series was designed "3’-deoxy" or "3’-H." The most active compound of this series, with an amine negatively charged, shows an IC50 against the enzyme of 8 µM and an EC50 of 2.6 µM in replicon assay. In addition, in collaboration with Ivan Barvik, we performed a structural model and we propose an interpretation of the gain provided by the absence of 3’-OH groups. Indeed, the absence of 3’-OH leads to a better stabilization of the Sp diastereomeric dinucleotide conformation in the catalytic site. In addition, the stabilization due to hydrogen bonds with the additional amino acid of the catalytic site of the enzyme affects the interactions between the oxygen atom present on the phosphorus opposite the phosphoramidate bond and catalytic magnesium ions, leading to additional stabilization. (See Figure 3).

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Figure 3
Molecular model of dinucleotide Sp/Rp GC 3’-H and Sp/Rp GC 3’-OH phosphoramidate in the active site of HCV NS5B polymerase

After the success obtained with dinucleotide phosphoramidate inhibitors, our project was to develop new series of "dinucleotides" GpC more flexible. WE designed PNA and chimera RNA/PNA. This work started in October 2011.
The PNAs are nucleic acid analogs in which the sugar-phosphate backbone of natural nucleoside is replaced by a pseudopeptide synthetic usually consisting of units of N-(2-aminoethyl)-glycine. In the RNA-PNA chimera, the junction is produced by phosphoramidate or phosphodiester linkage.

Publications

  • Priet S, Zlatev I, Barvik I, Geerts K, Leyssen P, Neyts J, Dutartre H, Canard B, Vasseur JJ, Morvan F, Alvarez K (2010) J Med Chem 53 6608-17
  • Zlatev I, Dutartre H, Barvik I, Neyts J, Canard B, Vasseur JJ, Alvarez K, Morvan F (2008) J Med Chem 51 5745-5757

Collaborateurs

- Drs Jean-jacques Vasseur et François Morvan, UMR 5625, CNRS, Université de Montpellier II, Laboratoire de Chimie Organique Biomoléculaire de Synthèse. Montpellier, France.
- Drs Johan Neyts et Jan Balzarini, Rega Institute for medical research, Université catholique de Leuven , Belgique.
- Pr Ivan Barvik Jr. Institute of Physics, Charles University, Ke Karlovu 5, Prague 12116 2, Republique Tcheque.

Financements

- Bourse doctorale du ministère (Septembre 2011 – 2014).
- Financement ANRS (2 ans), 115100 euros (janvier 2005 – 2007). En collaboration avec les Drs J.J. Vasseur et F. Morvan « des dinucléotides modifiés comme inhibiteurs potentiels du complexe réplicatif du virus de l’hépatite C ».

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