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Molecular Mechanism of HIV-1 Integrase Inhibition by Raltegravir Proposed by Using of Molecular Modeling Approaches Rohit Arora

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Molecular Mechanism of HIV-1 Integrase Inhibition by Raltegravir Proposed by Using of Molecular Modeling Approaches Rohit Arora Molecular mechanism of HIV-1 integrase inhibition by Raltegravir proposed by using of molecular modeling approaches Rohit Arora To cite this version: Rohit Arora. Molecular mechanism of HIV-1 integrase inhibition by Raltegravir proposed by using of molecular modeling approaches. Agricultural sciences. École normale supérieure de Cachan - ENS Cachan, 2012. English. NNT : 2012DENS0055. tel-00905951 HAL Id: tel-00905951 https://tel.archives-ouvertes.fr/tel-00905951 Submitted on 19 Nov 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ENSC - 2012n°394 THESE DE DOCTORAT DE L’ECOLE NORMALE SUPERIEURE DE CACHAN Présentée par Monsieur Rohit ARORA pour obtenir le grade de DOCTEUR DE L’ECOLE NORMALE SUPERIERE DE CACHAN Domaine : Sciences de la Vie et de la Santé Sujet de la thèse : Molecular mechanism of HIV-1 integrase inhibition by Raltegravir proposed by using of molecular modeling approaches Thèse présentée et soutenue à Cachan le 26 octobre 2012 devant le jury composé de : Philippe CUNIASSE Directeur de Recherche Rapporteur/President Florent BARBAULT Maitre des Conférences Rapporteur Liliane MOUAWAD Chargé de Recherche Examinatrice Luba TCHERTANOV Directrice de Recherche Directrice de thèse Nom du Laboratoire : Laboratoire de Biologie et Pharmacologie Appliquée ENS CACHAN/CNRS/UMR8113 61, avenue du Président Wilson, 94235 CACHAN CEDEX, France 0 Mots clefs : SIDA, Intégrase du VIH-1, ADN virale, inhibition des cibles, Raltegravir, modélisation, reconnaissance moléculaire, simulation de dynamique moléculaire, amarrage Le résumé L'intégrase (IN) rétrovirale est responsable de l’intégration de l'ADN viral du VIH -1dans l’ADN ce llulaire, processus indispensable à la réplication virale. Ce processus se déroule en deux étapes indépendantes, le 3’ -processing et le transfert de brins, catalysées par l’IN. La compréhension des interactions entre l’IN et l’ADN viral et de la cinétique de formation des complexes pré-intégratifs a permis l’identification du raltégravir (RAL) et de l’elvitégravir (ELV) qui se sont avérés être des inhibiteurs très efficaces de la réplication virale . Le RAL, auparavant désigné sous le code MK-0518, est un nouveau médicament anti-VIH qui a obtenu son autorisation de commercialisation aux Etats-Unis sous le nom de Isentress TM le 12 octobre 2007. Le ELV est toujours en essais cliniques. Toutefois, comme on l'observe pour d'autres antirétroviraux, ces composés n’échappent cependant pas aux phénomènes de résistance. Des mutations de résistance spécifiques au RAL ont ainsi été identifiées chez des patients. À ce jour, aucune donnée expérimentale caractérisant la structure de l’IN du VIH -1, la structure au RAL et/ou les interactions du RAL avec sa cible n'a été rapporté. Premièrement, nous avons caractérisé les propriétés structurales et conformationnelles du RAL dans des états différents, en phase gazeuse, en solution dans l'eau et à l'état solide. Une etude détaillée a permis de caracteriser la reconnaissance du RAL par des cibles virals, l’IN et l’ADN viral avant et après la réaction de 3’ -processing. Nous avons trouvé que le RAL adopte un large spectre de conformations et configurations dans des états isolés et/ou liés avec le(s) cible(s). Les meilleures score et poses de docking confirment que le modèle représentant le complexe IN•vADN est la cible biologiquement pertinente du RAL. Ce résultat est cohérent avec le mécanisme d'inhibition du RAL communément admise. Nous avons suggéré que le processus d'inhibition peut comprendre dans un premier temps la reconnaissance du RAL par l'ADN viral clivé et lié à un état intermédiaire de l’IN. Le RAL couplé à l’ADN viral montre une orientation à l'extérieur de tous les atomes d'oxygène, d'excellents agents putatifs pour capturer de cations Mg 2+ , ce qui pourrait faciliter l'insertion du RAL dans le site actif. La flexibilité conformationnelle du RAL permet l'adaptation de l'inhibiteur dans une poche relativement grande de du complexe IN•vADN, permettant la production de diverses conformations du RAL. Nous croyons que cette diversité des conformations du RAL contribue à la reconnaissance de résidus enzymatiques et peut influer sur le choix des voies alternatives de résistance au RAL observées cliniquement. Nous avons étudié également la reconnaissance par l’IN des inhibiteurs du VIH appartenant à différents souches, B et CRF02_AG. Nous avons montré que la structure de l’IN des deux souches est quasi -identique. Le docking du RAL et de deux autres inhibiteurs de transfert de brins (ELV et L731_988) sur chaque modèle montre que leur reconnaissance par deux différentes souches cibles est identique. Notre analyse des effets moléculaires et structuraux des mutations de résistance sur la structure de l’IN a montré que les structures de l’enzyme sauvage et mutante sont aussi quasi - identique. Par contre, les mutations modifient considérablement la spécificité de reconnaissance de l'ADN par l'IN. Nous avons eff ectué la simulation de dynamique moléculaire (MD) de l’IN sauvage et mutant, avec une mutation ponctuelle R228A localisée dans le domaine C-terminale. Notre étude de la flexibilité de l’IN et du complexe IN•ADN par la dynamique moléculaire ouvre une voie très prometteuse non seulement sur le plan de la recherche fondamentale mais aussi pour l'application de nos concepts au développement de nouvelles générations d'inhibiteurs ciblant l'IN. 1 Keywords : AIDS, HIV-1 Integrase, viral DNA, targets inhibition, Raltegravir, modeling, molecular recognition, molecular dynamics simulation, docking Summary The HIV-1 integrase catalyzes the integration of HIV-1 viral DNA (vDNA) into the host cell chromosome in a process, which is essential for viral replication through two independent reactions, 3’ -processing (3’ -P) and strand transfer (ST), catalyzed by IN. Deciphering the structural determinants of the interaction between integrase and its substrates and the kinetics of this interaction sheds light on the importance of inhibitors targeting the pre-integration IN•vDNA complex. This approach led to the identification of raltegravir (RAL) and elvitegravir (ELV), which turned out to be highly efficient inhibitors of ST. RAL, formerly known under the code MK-0518, is a new anti-HIV drug that obtained clinical approval in the United States under the name Isentress TM on October 12, 2007. ELV is still in clinical trials. However, these compounds nevertheless encounter resistance phenomenon. To date, no experimental data characterizing the RAL structure, structure of the HIV-1 IN and/or interactions of RAL with its targets, has been reported. First, we characterized the structural and conformational properties of RAL in different states ‒ the gas phase, in water solution and the solid state. Second, a detailed study allowed characterisation the RAL recognition by the viral targets ‒ IN and the vDNA, before and after the 3'-P. We found that RAL shows a broad spectrum of conformations and configurations in isolated state and/or associated with the target(s). The best docking poses and scores confirmed that the model representing IN•vDNA complex is a biologically relevant target of RAL. This result is consistent with the commonly accepted mechanism of RAL inhibition. Based on the docking results we suggested that the inhibition process may include, as a first step, the RAL recognition by the processed vDNA bound to a transient intermediate IN state. RAL coupled to vDNA shows an outside orientation of all oxygen atoms, excellent putative chelating agents of Mg 2+ cations, which could facilitate the insertion of RAL into the active site. The conformational flexibility of RAL further allows the accommodation/adaptation of the inhib itor in a relatively large binding pocket of IN•vDNA pre-integration complex thus producing various RAL conformation. We believe that such variety of the RAL conformations contributing alternatively to the enzyme residue recognition may impact the selection of the clinically observed alternative resistance pathways to the drug. We also studied the recognition of the HIV-1 IN inhibitors from two different strains, B and CRF02_AG. Our in silico study showed that the sequence variations between CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. Our analysis of the resistance mutations effects showed that structure of the wild-type enzyme and mutants is almost identical. However, the resistance mutations significantly altered the specificity of the viral DNA recognition by IN. We performed molecular dynamics simulations of the native and mutated IN with a point mutation R228A localized in the C-terminal domain. The study of targets flexibility opens a very promising way, not only in terms of fundamental research, but also for the application of our concepts to the development of new generations of inhibitors targeting IN. 2 Acknowledgements Working on my Ph.D. has been a wonderful and often overwhelming experience. It is hard to say whether is has been grappling with the topic itself which has been the real learning experience, or grappling with how to write papers, give talks and work in a group. In any case, I am indebted to many people for making the time working on my Ph.D. an unforgettable experience. I am grateful to Doctors Phillipe Cuniasse and Florent Barbault for accepting to be the reviewers of my thesis. Their comments and critique have been extremely helpful in improving my thesis and making it a rather learning experience.
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