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 cellulaire, 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 IsentressTM 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 Mg2+, 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 effectué 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 IsentressTM 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 Mg2+ cations, which could facilitate the insertion of RAL into the active site. The conformational flexibility of RAL further allows the accommodation/adaptation of the inhibitor 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. I sincerely thank Doctor Liliane Mouwad who accepted to examine my work. Words cannot describe my gratitude to my thesis supervisor, Doctor Luba Tchertanov, for welcoming me to her team BiMoDyM at LBPA. She patiently guided me through my master’s project – which was a relatively new field for me – and helped me prepare for my PhD thesis. We have had many fruitful discussions and brainstorming sessions over the last 4 years. Her outstanding ability, scientific and pedagogical, together with personal kindness and openness turned my work into truly one of a kind experience. Whatever she does, she does it with taste and elegance and I hope I was able to absorb at least a little bit of these qualities. My sincere gratitude is reserved for all the current and former members of my group, for making it a memorable experience. Especially - Elodie Laine, Isaure Chauvot de Beauchene, Safwat Abdel-Azeim and Joseph Andre – who contributed in one way or the other to my research. This work would not have been possible without the great number collaborators. I am much obliged to Jean-Christophe Lambry (Ecole Polytechnique), Marina Gottikh (Moscow State University), the team of Jean-Francois Mouscadet (LBPA, ENS de Cachan) and clinicians from the Hospital Pitié-Salpetriere (Paris). A special mention to companies - Schrodinger, Tripos and CCDC - who helped in providing licenses and maintenance of some key softwares used in my research. A special thanks to Florent Langenfeld for his patience in correcting my oral and written French. I am also indebted to the ENS Cachan for giving me an opportunity to conduct my PhD thesis at this prestigious institute. I am grateful to the Ecole Doctorale des Sciences Pratiques (EDSP) de Cachan and the French Ministry of Higher Education for providing and managing the financial support for my PhD research during a period of three years. I am extremely grateful to Professor Isabelle Ledoux-Rak who introduced me to ENS Cachan and LBPA, and provided support at every step during my Master’s program as well as during the application process of my PhD. I am also very thankful to all the professors of my Master’s program for providing me with excellent guidance and education. Finally, I thank my parents and my family without whom none of this would have been possible. I thank my dear friends – Hillary Kloeckner, Dhruv Shah and Karthik Aluru – who provided me with support and encouragement whenever I needed it the most. They have been my family away from home. 3 Content ABBREVIATIONS ......................................................................................... 8 Chapter 1. INTRODUCTION ................................................................. 9 I. HIV, AIDS and Antiretroviral Therapy..................................................... 9 1. HIV epidemiology and polymorphism...................................................... 9 2. Human Immunodeficiency Virus Type 1.................................................. 14 3. HIV-1 subtypes and circulating recombinant forms.................................. 16 4. HIV replication cycle................................................................................. 18 II. Antiretroviral Drugs...................................................................................... 20 1. Entry and Fusion Inhibitors....................................................................... 20 2. The Reverse Transcriptase Inhibitors (RTIs)............................................. 21 3. The Protease Inhibitors (PIs)...................................................................... 22 4. The Integrase Inhibitors (INIs) .................................................................. 23 5. Highly Active Antiretroviral Therapy (HAART) and resistance effect to drugs. ........................................................................ 24 III. Structure and Functions of HIV-1 Integrase.............................................. 26 1. Structural characterization of the HIV-1 Integrase.................................. 26 1.1 Experimental Data.............................................................................. 26 1.2 Theoretical Models............................................................................. 29 1.3. Structural and functional role of the catalytic site loop..................... 31 1.4 Molecular Dynamics Simulation of HIV-1 Integrase........................ 34 2. HIV-1 Integrase functions........................................................................ 36 2.1 Integrase activity................................................................................. 36 2.2 Role of the cationic co-factors............................................................ 37 2.3 Mechanisms of inhibition and target-inhibitors interactions.............. 38 3. Resistance to Integrase Strand Transfer Inhibitors (INSTIs)..................... 46 3.1 Resistance phenomenon...................................................................... 46 3.2 Polymorphism effect........................................................................... 50 IV. Raltegravir-the first clinically used integrase specific drug..................... 51 1. Discovery and development of Raltegravir............................................. 51 2. Efficiency of Raltegravir......................................................................... 52 2.1 Antiviral activity in vivo.................................................................... 52 2.2 Safety................................................................................................. 53 2.3 Pharmacokinetics............................................................................... 54 3. Viral resistance to Raltegravir.................................................................. 55 V. Molecular Modeling Approaches................................................................. 56 1. Ab-initio Methods..................................................................................... 58 4 2. Fragment-Based Structure Analyses........................................................ 58 3. Docking.................................................................................................... 60 3.1 Protein-Ligand Docking..................................................................... 60 3.2 Protein-DNA Docking........................................................................ 61 4. Molecular Dynamics Simulations............................................................ 62 5. Homology Modeling................................................................................. 63 Chapter 2. RESULTS ..................................................................................... 65 I. Raltegravir conformations in gas, water solution and solid state............ 65 1. Raltegravir conformations in the gas phase............................................... 66 2. Molecular dynamic simulations of Raltegravir in water solution............... 68 3. Fragment-based structural analysis............................................................. 70 3.1 Raltegravir configurational/conformational properties........................ 70 3.2 Probing of the Raltegravir coordination to biologically relevant cations - Mg, Mn and K.......................................................... 75 4. Discussion................................................................................................... 79 II. Targets models, representing the HIV-1 Integrase and viral DNA before and after 3’-processing..................................................................... 81 1. Modelling of the biologically relevant HIV-1 targets.............................. 81 1.1 Models of the unbound Integrase....................................................... 81 1.2 Models of the IN●vDNA complex..................................................... 83 III. Raltegravir – Targets recognition............................................................. 86 1. Docking poses and conformations............................................................ 86 2. Evaluation of docking algorithms............................................................. 87 3. The viral DNA as a putative Raltegravir target......................................... 90 4. Discussion.................................................................................................. 92 IV. Comparison of Integrase structure from the HIV-1 subtypes B and CRF02_AG and its susceptibility to Integrase Strand Transfer Inhibitors........................................................................ 96 1. Structural analysis of Integrase from B and CRF02_AG strains.............. 96 2. The INSTIs recognition by Integrase from B and CRF02_AG strains.... 98 3. Discussion.................................................................................................. 102 V. Alternative molecular recognition of DNA induced by Raltegravir resistance mutations..................................................................................... 106 1. Evidence of a stable W-shaped hairpin in the catalytic site loop................ 106 5 2. Modeling of the catalytic site structure of the HIV-1 integrase.................. 108 3. Effect of Raltegravir-selected mutations on Catalytic Core Domain structure....................................................................................................... 112 4. Model of the displacement of the W-shaped hairpin towards the catalytic site................................................................................................. 114 5. Intermolecular interactions of the Raltegravir-selected mutated residues.......................................................................................... 115 6. 3D maps of H-bonding between the residues 148 and 155 and DNA bases............................................................................................. 117 6.1 The DNA bases recognition by the Wild Type Integrase residues N155 and Q148......................................................................... 118 6.2 The DNA bases recognition by the mutated Integrase residues N155H and Q148R/H/K.......................................................................... 119 7. Modeling the ‘interacting’ DNA base pairs.................................................. 121 7.1 Arginine side chain interactions with A–T and G–C base pairs............. 121 8. Discussion..................................................................................................... 123 VI. Molecular Dynamics Simulation of the unbound Integrase in the native and mutated form.............................................................................. 126 1. Root Mean Squared Distances (RMSD) Comparisons............................... 127 2. Root Mean Squared Fluctuations (RMSF) Analysis.................................. 129 3. Principal Component Analysis (PCA)....................................................... 130 3.1 Trajectory Analysis.............................................................................. 131 3.2 Eigen RMSFs Analysis........................................................................ 132 4. Structural Analysis..................................................................................... 133 5. Secondary Structure Analysis.................................................................... 137 6. Discussion.................................................................................................. 140 Chapter 3. GENERAL CONCLUSIONS AND PERSPECTIVES ................................................................. 141 Chapter 4. MATERIALS AND METHODS .............................. 145 I. Probing of Raltegravir structure.................................................................. 145 1. Conformational analysis............................................................................. 145 2. Molecular Dynamics Simulations.............................................................. 145 3. Structural fragment-based analysis (Cambridge Structural Database)....... 146 II. Targets modeling........................................................................................... 147 1. The HIV-1 Integrase models of the B and CRF02_AG strains................ 147 2. IN•DNA Models of the B and CRF02_AG strains.................................. 148 3. Secondary structure prediction.................................................................. 149 4. Generation of the models of unbound IN , IN •vDNA complex HIV HIV PFV and vDNA ............................................................................................. 150 HIV 6 III. Molecular Docking Protocols........................................................................ 151 1. Integrase Strand Transfer Inhibitors (INSTIs) binding with IN and IN•vDNA complex of B and CRF02_AG strains...................................... 151 2. Raltegravir docking onto the targets-I , I •vDN complex and NHIV NHIV APFV vDNA ................................................................................................... 152 HIV IV. Molecular Dynamics Simulations of unbound Integrase........................... 154 V. Molecular Modelling..................................................................................... 155 1. Wild type IN models preparation.............................................................. 155 2. Model minimizations................................................................................. 156 3. Simulations of the movement of the 140–149 loop towards the catalytic site.......................................................................................... 156 4. Characterization of the side-chains and DNA bases interactions.............. 157 REFERENCES .................................................................................................. 159 APPENDIX ........................................................................................................... 178 7 ABBREVIATIONS 2D In two dimensions 3D In three dimensions 3’-P 3’-Processing Reaction AIDS Acquired Immuno-deficiency Syndrome ARV Anti-retroviral CCD Catalytic Core Domain CTD C-Terminal Domain DNA Deoxyribonucleic Acid ELV Elvitagravir GLIDE Grid-based Ligand Docking with Energetics HIV Human Immuno-deficiency Virus INSTI Integrase Strand Transfer Inhibitors LEDGF Lens epithelium-derived growth factor MD Molecular Dynamics NMR Nuclear Magnetic Resonance NTD N-Terminal Domain PCA Principal Component Analysis PDB Protein Data Bank PFV Primate Foamy Virus PIC Pre-Integration Complex RAL Raltegravir RMSD Root Mean Squared Distance RMSF Root Mean Squared Fluctuations RNA Ribonucleic Acid ST Strand Transfer Reaction WT Wild Type 8 Chapter 1. INTRODUCTION I. HIV, AIDS and Antiretroviral Therapy 1. HIV Epidemiology and Polymorphism In 1981, early cases of a new human epidemic began to emerge in the United States of America. In June 1981, the Centre for Disease Control and Prevention (CDC) published an article in Morbidity and Mortality Weekly Report which reported that 5 homosexual men were diagnosed with Pneumocystis carinii pneumonia in Los Angeles (Centers for Disease Control, 1981a). Soon after this report, another article published by CDC reported the cases of Kaposi’s Sarcoma among 26 homosexual men in the United States of America - 20 in New York and 6 in Los Angeles (Centers for Disease Control, 1981b). These reports were the first official reporting of a disease that would later become known as the AIDS epidemic. The CDC published its first definition of AIDS in September 1982 as “a disease, at least moderately predictive of a defect in cell mediated immunity, occurring in a person with no known cause for diminished resistance to that disease”(Centers for Disease Control, 1982). It was characterized by diseases resulting from an impaired immune system. In 1978, Robert C. Gallo’s group reported the discovery of the first human retrovirus, Human T-cell Leukemia Virus Type 1 (HTLV-I) (Gallo et al., 1978). HTLV-I was the first of the only four retroviruses that infect human beings (the other three being HTLV-II, HIV-1 and HIV-2). HTLV-I is a retrovirus containing single-stranded RNA and causes cancers such as T-cell leukemia and T-cell lymphoma in adults. Gallo’s team and his collaborators had also discovered the first cytokine called T-Cell Mitogenic factor, which was later named as Interleukine-2 (IL-2) in 1976 (Morgan, Ruscetti, & Gallo, 1976) which was the to be identified. IL-2 is a vital growth factor for the T-lymphocytes in the presence of which the 9