LABORATOIRE de BIOLOGIE et PHARMACOLOGIE APPLIQUEE (LBPA-CNRS), ENS de CACHAN, FRANCE

1. XVIII International AIDS Conference Vienna, 18-23 July 2010 THPDA201 IMPACT OF THE HIV-1 INTEGRASE FLEXIBILITY ON THE ALTERNATIVE RESISTANCE PATHWAYS TO RALTEGRAVIR S. Abdel-Azeim, E. Laine, I. Chauvot de Beauchêne, D. Perahia, J.-F. Mouscadet and L.Tchertanov TM Raltegravir (Isentress) Malet et al., Antimicrob. Agents&Chemother. 2008; Delelis et al., NucleicAcidsRes. 2009; Mouscadet et al., J. Mol. Rec. 2009; Mouscadet et al., Drug Resist. Updates, 2010 LABORATOIRE de BIOLOGIE et PHARMACOLOGIE APPLIQUEE (LBPA-CNRS), ENS de CACHAN, FRANCE e-mail: luba.tchertanov@lbpa.ens-cachan.fr

2. INTEGRASE MODELS and their FLEXIBILITY 3’-processing • Molecular Dynamics • Normal Modes Analysis Mg2+ Strand Transfer Mg2+ 2 Mg2+ +Ve +Ve -Ve -Ve

3. COMPARATIVE ANALYSIS of the WT and MUTATED INTEGRASE The energy of conformations produced by the NMs as a function of the displacement for the WT and Y143R mutant. A In the IN (Apo) RAL binding site depends on the loop conformation (open/close) B The Ral docking into IN•DNA proved that the presence of DNA is required for RAL to bind efficiently The RAL and DNA bases recognition by IN•Mg: 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. 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

4. Conclusions and Perspectives • ♪Weappliedtheoreticalmethods to study the origin of the alternative resistancepathwaysselected by Ral; • ♪Weconstructed the modelscorresponding to 3’–processing and ST pre-integrationcomplex; • ♪Weverifiedthreehypothesiswhichcanlead to the differentchoice of the resistacepathways: • (i) the effect of the naturalpolymorphism; • (ii) the role of potential cis-effect of the reverse transcriptase on the integrase; • (iii) the targets and inhibitorflexibility. • ♫ Wefoundthat the last effectisdecisive in the RAL-Target recognition process, particularly for the RAL poses while the bindingenergies are similar for the differentloop 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. • ♪Presentlywe are constructing the inhibition steps in the « real time » to follow the wholeprocess (Molecular Dynamics Simulations

5. In silicoModelisation Bioinformatics, Molecular Dynamics and Modelisation (BiMoDyM) L. Tchertanov,Research Director D. Perahia, Research Director E. Laine, Post Doc S. Abdel-Azeim, Post Doc M. Lopez-Ramos, Post Doc P. Ricardo Batista, Post Doc D. Enry Gomes, Post Doc R. Arora, PhDstudent I. Chauvot de Beauchene, PhDstudent Biochemistry Retrovirology J.-F. Mouscadet, Research Director O. Delelis, ResearchScientist F. Subra, ResearchScientist S. Thierry, Post Doc X.-J. Ni, PhDstrudent ClinicalStudies Hôpital Pitié-Salpêtrière Virology, INSERM V. Calvez, Professor I. Malet, Dr A.-G. Marcelin, Dr M.-A. Valantin C. Soulie, A. Derache, M. Wirden C. Katlama