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Development of new phosphonate nucleoside analogs to circumvent the resistance of HIV-1 RT against nucleotides.

Head Karine ALVAREZ

Introduction

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is essential in the process of viral replication and therefore represents an obvious target of choice for antiviral products. The nucleoside is an essential part of current antiretroviral therapy. Unfortunately, treatments using nucleoside analogues lose their effectiveness over time, while simultaneously resistant viruses emerge. The abundance of HIV-1 strains resistant to multiple antiviral drugs makes the current situation of great concern.

Our goal is to design a new generation of nucleotide inhibitors that are more efficient than current antiretroviral therapy used to block HIV-1 RT irreversibly.

Our group is interested very closely to these resistance problems. Their understanding is essential to guide us to design new and better antivirals. The Tenofovir prodrug (VIREAD®, Gilead) was approved by the FDA in October 2001 as a single agent against HIV-1 has received marketing authorization in Europe in February 2002. This is a major drug in the fight against HIV. This compound is a nucleotide analog type acyclic phosphonate. It has revolutionized the treatment against HIV-1 and has really improved decision (frequency, dose) of the drug. However, like all other nucleosides used in therapy, viruses (especially those already resistant to AZT) respond poorly to treatment or become resistant to this compound (K65R).

boranophosphonates

People involved : Dr Karine Alvarez, Dr Karine Barral, Dr Céline De Michelis, Dr Antoine Frangeul.

In 2002, we have developed new nucleotide analogues: the α-boranophosphonates, designed to achieve a synergy of biochemical and enzymatic properties of “borano” modification and phosphonate derivatives. The first α-boranophosphonates derivatives of AZT, d4T, PMPA and PMEA were synthesized using an original method.

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Figure 1
Structure of target boranophosphonates

The compounds were evaluated in infected cell cultures with various viruses (HIV-1 and 2), and none showed activity, or toxicity. To explain the inactivity of these boranophosphonates, we attempted to answer the following questions:
- Are these compounds are stable in biological fluids?
- Are they phosphorylated by cellular kinases and human hAK1 hAK2?
- Are they good substrates for the reverse transcriptase of HIV-1? To the question of their stability, we may have answered that these compounds degrade relatively quickly in complete culture medium and cell extracts for some of them, with half-life of less than 3 hours. Degradation of BH3- analogues is due to a reduction in binding P-B to P-H bond, leading to the formation of H-phosphinates corresponding particularly stable in fluids biological, but also inactive in infected cell cultures. We were also able to show that they were very poorly phosphorylated by kinases and human hAK1 hAK2, with catalytic efficiencies, lower more than 105 times those of the corresponding natural analogues.

Finally, kinetic studies in pre-steady state, were performed with of BH3-PMEApp and BH3-PMPApp. We demonstrated that these compounds reduce or circumvent the resistance of the K65R mutant RT compared to similar non borano PMEApp and PMPApp. Boranophosphonates compounds are unfortunately not applicable in chemotherapy against HIV.

Publications

  • Barral K, Priet S, De Michelis C, Sire J, Neyts J, Balzarini J, Canard B, Alvarez K (2010) Eur J Med Chem 45 849-56
  • Topalis D, Alvarez K, Barral, Munier-Lehmann H, Schneider B, V�ron M, Guerreiro C, Mulard L, El-Amri C, Canard BDeville-Bonne D (2007) Nucleosides Nucleotides Nucleic Acids 27 319-31
  • Frangeul A, Busseta C, Deval J, Barral K,Selmi B, Boretto J, Alvarez K, Canard B (2008) Antivir Ther 13 (1) 115-24
  • Frangeul A, Barral K, Alvarez K, Canard B (2007) Antimicrob Agents Chemother 51 3162-7
  • Barral K, Priet S, Sire J, Neyts J, Balzarini J, Canard B, Alvarez K (2006) J Med Chem 49 7799-7806
  • Deval J, Alvarez K, Selmi B, Bermond M, Boretto J, Guerreiro C, Mulard L, Canard B (2005) J Biol Chem 280 3838-46
  • Alvarez K, Deval J, Selmi B, Barral K, Boretto J, Guerreiro C, Mulard L, Sarfati R, Canard B (2005) Nucleosides Nucleotides Nucleic Acids 24 419-21

Collaborators

- Drs Johan Neyts / Jan Balzarini, Rega Institute for medical research , Université catholique de Leuven , Belgique.
- Dr Dominique Deville-Bonne.
- Dr Joséphine Sire.
- Pr Fabien Zoulim, Faculté de médecine RTH Laennec, Lyon.

Fundings

- Bourse post-doctorale SIDACTION pour le Dr C. De Michelis (janvier 2003 - janvier 2005)
- Bourse post-doctorale ANRS pour le Dr K. Barral (janvier 2004 - janvier 2006)
- ESC-Sidaction. Projet intitulé « conception de nouveaux inhibiteurs nucléotidiques dirigés contre la RT du VIH-1 : contourner la résistance aux drogues de type nucléosidique». 50000 euros sur 2 ans.

Thiophosphonates : Adethiovir and Tenothiovir.

People involved: Dr Karine Alvarez, Dr Karine Barral, Dr Antoine Frangeul, Nadine Payrot, Dr Stéphane Priet, Loic Roux, Clément Weck.

We have discovered thiophosphonates active against HIV-1, HIV-2 and HBV-infected cells culture. These compounds were baptized Adethiovir or S-PMEA and Tenothiovir or S-PMPA .

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Figure 2
Structure of thiophosphonates

Ces composés sont couverts par une demande de brevet de notre part (B. Canard, K. Alvarez, K. Barral, J.L. Romette, J. Neyts, J. Balzarini, WO/2008/056264, (2008)).

These compounds are patented B. Canard, K. Alvarez, K. Barral, J.L. Romette, J. Neyts, J. Balzarini, WO/2008/056264, (2008). They show excellent antiviral activity similar to that of adefovir and tenofovir, and show no cytotoxicity. Their preliminary pharmacology is favorable: they are stable over 24 hours in media mimicking biological fluids (culture medium and cell extracts). We synthesized the corresponding diphosphates S-PMEApp and S-PMPApp. Kinetic studies in pre-steady state allowed us to demonstrate that these compounds are substrates and inhibitors of HIV-1 RT wild type and mutant K65R. They are incorporated and chain terminators. In addition, the S-PMPApp eliminates the resistance of the K65R mutant RT. Thus, the IC50 is found to be similar or even better, the affinity of these compounds is improved and the initial rate of excision of these compounds by the AZT-resistant RT is decreased by a factor of 7-18. This is due to the fact that our compounds block the RT in an inert conformation. These derivatives thiophosphonates therefore have a significant added value compared to adefovir and tenofovir, including the fight against the resistance mutants selected by current therapies.

We have synthesized bioavailable forms. They showed improved antiviral activity, validating the prodrug strategy applied to our compounds. Thiophosphonates Adethiovir and Tenothiovir have already passed some important milestones of their characterization. These preliminary results make these molecules as promising drug candidates. These compounds could offer the same benefits as tenofovir or Adefofir, but with better ability to target resistant viruses and probably a different spectrum of resistance.

Publications

- Barral K, Weck C, Payrot N, Roux L, Durafour C, Zoulim F, Neyts J, Balzarini J, Canard B, Priet S, Alvarez K (2011) Eur J Med Chem 46 4281-8

- Patent B. Canard, K. Alvarez, K. Barral, J.L. Romette, J. Neyts, J. Balzarini, WO/2008/056264, (2008)

Collaborateurs

- Drs Johan Neyts / Jan Balzarini, Rega Institute for medical research , Université catholique de Leuven , Belgique.
- Dr Jean-luc Galzi, IFR 85 Gilbert Laustriat, Biomolécules et innovations thérapeutiques, Plate-forme « Preclinical Drug Technology », Faculté de Pharmacie et Ecole Supérieure de Biotechnologie, ILLKIRCH Cedex
- Pr Fabien Zoulim, INSERM, U871, Université de Lyon, Hospices Civils de Lyon, 69003.
- Pr Vincent Calvez. Service de Virologie Hopital Pitié-Salpêtrière 83 Bd de l’hôpital 75013 Paris.

Fundings

- Bourse post-doctorale ANRS (2 ans), 2010–2012, pour le Dr Stéphane Priet. « Tenothiovir et adethiovir: nouveaux analogues phosphonates acycliques pour cibler les VIH-1 resistants ».
- Financement ANRS (2 ans), 150000 euros (septembre 2007 – septembre 2009). « Tenothiovir et adethiovir: nouveaux analogues phosphonates acycliques pour cibler les VIH-1 resistants ». Financement d’une prestation de service auprès de la société SPI-BIO pour caractériser les thiophosphonates dans un contexte cellulaire (virus sauvage et mutants cliniquement relevants).
- Complément de Demande d’Aide aux Equipes (SIDACTION), obtenue en mai 2007. « Découverte de nucléotides thiophosphonates actifs contre le VIH. Evaluation sur virus résistants » N/Ref : 10016-02-00/AO17-2. Salaire Ingenieur d’Etude, 1 an, Nadine Payrot (juin 2007 – mai 2008).
- Demande d’Aide aux Equipes (SIDACTION), obtenue en juillet 2006. « Découverte de nucléotides thiophosphonates actifs contre le VIH. Evaluation sur virus résistants » N/Ref : 10016-02-00/AO17-2. 25000 euros sur 1 an (Nov 2006 – Nov 2007)

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