LABORATOIRE de BIOLOGIE et PHARMACOLOGIE APPLIQUEE (LBPA-CNRS), ENS de CACHAN, FRANCEe-mail: luba.tchertanov@lbpa.ens-cachan.fr

S. Abdel-Azeim, E. Laine, I. Chauvot de Beauchêne, D. Perahia, J.-F. Mouscadet and L.Tchertanov

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IMPACT OF THE HIV-1 INTEGRASE FLEXIBILITY ON THE ALTERNATIVE RESISTANCE PATHWAYS TO RALTEGRAVIR

N N

O

HN

O

O

N N

H3C

H3C

O

HN

F

OH

Raltegravir (Isentress )

XVIII International AIDS Conference Vienna, 18-23 July 2010THPDA201

TM

Malet et al., Antimicrob. Agents&Chemother. 2008;Delelis et al., Nucleic Acids Res. 2009; Mouscadet et al., J. Mol. Rec. 2009;Mouscadet et al., Drug Resist. Updates, 2010

3’-processing

Strand Transfer

-Ve

+Ve 2 Mg2+

-Ve

+Ve

Mg2+

Mg2+

INTEGRASE MODELS and their FLEXIBILITY

Molecular Dynamics Normal Modes Analysis

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The energy of conformations produced by the NMs as a function of the displacement for the WT and Y143R mutant.

The RAL and DNA bases recognition by IN•Mg:

(A) Binding energies as a function of the displacement for the WT and Y143R mutant. The docking energy does not show significant difference between the distinct conformations.

(B) Binding energies for the WT, Y143R, Q148R and N155H mutants with IN and IN•DNA.

PM3/CHARMM optimized RAL poses in the WT, Y143R , Q148R and N155H INs

COMPARATIVE ANALYSIS of the WT and MUTATED INTEGRASE

A

B

The Ral docking into IN•DNA proved that the presence of DNA is required for RAL to bind efficiently

In the IN (Apo) RAL binding site depends on the loop conformation (open/close)

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Conclusions and Perspectives

- ♪ We applied theoretical methods to study the origin of the alternative resistance pathways selected by Ral;

- ♪ We constructed the models corresponding to 3’–processing and ST pre-integration complex;

- ♪ We verified three hypothesis which can lead to the different choice of the resistace pathways: (i) the effect of the natural polymorphism; (ii) the role of potential cis-effect of the reverse transcriptase on the integrase; (iii) the targets and inhibitor flexibility. - ♫ We found that the last effect is decisive in the RAL-Target recognition process, particularly for

the RAL poses while the binding energies are similar for the different loop conformations ;

- ♫ During or after the 3’-processing the viral DNA induces a structural modification prompting a movement of the loop towards the catalytic site which allows the fixation of the second Mg cation. The resulting pre-integration complex IN (2Mg)•DNA with the flexible unpaired –CA bases is a target for the ST inhibitor binding.

-♫ We postulated that during the inhibition process, Ral binds specifically to the Ade, displacing the -CA bases to adjust to the binding cavity and adapting its own conformation to coordinate with two Mg cations, thereby blocking the hydroxy group for the ST reaction. Slipping of Raltegravir into the cavity might be initiated by a specific interaction of the drug with the loop residues (148 or 143) when the loop folds over the active site.

♪ Presently we are constructing the inhibition steps in the « real time » to follow the whole process (Molecular Dynamics Simulations

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Biochemistry Retrovirology

J.-F. Mouscadet, Research Director O. Delelis, Research ScientistF. Subra, Research ScientistS. Thierry, Post DocX.-J. Ni, PhD strudent

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In silico ModelisationBioinformatics, Molecular Dynamics and Modelisation (BiMoDyM)

L. Tchertanov, Research Director D. Perahia, Research Director E. Laine, Post DocS. Abdel-Azeim, Post DocM. Lopez-Ramos, Post DocP. Ricardo Batista, Post DocD. Enry Gomes, Post DocR. Arora, PhD studentI. Chauvot de Beauchene, PhD student

Clinical StudiesHôpital Pitié-SalpêtrièreVirology, INSERM

V. Calvez, Professor I. Malet, Dr A.-G. Marcelin, Dr M.-A. Valantin C. Soulie, A. Derache, M. Wirden C. Katlama