DISSERTATIONES TECHNOLOGIAE UNIVERSITATIS TARTUENSIS 21 ANASTASIA SELYUTINA When biologist meets chemist: a search for HIV-1 inhibitors Tartu 2015 ISSN 2228-0855 ISBN 978-9949-32-972-4 DISSERTATIONES TECHNOLOGIAE UNIVERSITATIS TARTUENSIS 21 DISSERTATIONES TECHNOLOGIAE UNIVERSITATIS TARTUENSIS 21 ANASTASIA SELYUTINA When biologist meets chemist: a search for HIV-1 inhibitors Institute of Technology, Faculty of Science and Technology, University of Tartu, Estonia This dissertation is accepted for the commencement of the degree of Doctor of Philosophy in Biomedical Technology on 31th of August, 2015 by the Scientific council of the Institute of Technology, Faculty of Science and Technology, University of Tartu, Estonia Supervisor: Andres Merits, PhD, Professor of Applied Virology, Institute of Technology, University of Tartu, Estonia Reviewer: Irja Lutsar, MD, PhD, Professor in Clinical Microbiology, Institute of Microbiology, University of Tartu, Estonia Opponent: Anders Vahlne, MD, PhD, Professor of Clinical Virology and senior consultant at Karolinska Institutet and Karolinska University Hospital, Solna, Stockholm, Sweden Commencement: Auditorium 121, Nooruse 1, Tartu on 25th of November 2015 at 14.15 This research is supported by European Social Fund’s Doctoral Studies and Internationalisation Programme DoRa, which is carried out by Foundation Archimedes. ISSN 2228-0855 ISBN 978-9949-32-972-4 (print) ISBN 978-9949-32-973-1 (pdf) Copyright: Anastasia Selyutina, 2015 University of Tartu Press www.tyk.ee CONTENTS CONTENTS ................................................................................................ 5 LIST OF ORIGINAL PUBLICATIONS .................................................... 7 LIST OF ABBREVIATIONS ..................................................................... 8 INTRODUCTION ....................................................................................... 11 LITERATURE REVIEW ............................................................................ 13 1. VIRUS .................................................................................................... 14 1.1 History of discovery ....................................................................... 14 1.2 Origin and classification of HIV-1 ................................................. 14 1.3 Structure of the mature HIV-1 virion ............................................. 15 1.4 HIV-1 genome organization ........................................................... 16 1.5 HIV-1 infection cycle ..................................................................... 18 1.5.1 Cell binding and entry .......................................................... 18 1.5.2 Early post-entry events ......................................................... 18 1.5.3 HIV-1 cDNA integration ...................................................... 19 1.5.4 HIV-1 latency ....................................................................... 20 1.5.5 Transcription and transport of viral mRNAs to the cytoplasm........................................................................ 20 1.5.6 Translation of HIV-1 mRNAs .............................................. 21 1.5.7 Assembly, budding and maturation of HIV-1 virions .......... 22 2. REVERSE TRANSCRIPTASE AND REVERSE TRANSCRIPTION OF HIV-1 ............................................................................................... 24 2.1 History of discovery ....................................................................... 24 2.2 Biogenesis of HIV-1 reverse transcriptase ..................................... 24 2.3 Structure of HIV-1 reverse transcriptase ........................................ 25 2.4 The process of reverse transcription ............................................... 27 2.5 Molecular mechanisms used by reverse transcriptase and its polymerization reaction cycle ......................................................... 30 2.6 RNase H activity of HIV-1 reverse transcriptase ........................... 31 2.6.1 Structure ............................................................................... 31 2.6.2 Enzyme activity .................................................................... 31 2.6.3 Modes of RNase H cleavage ................................................ 32 2.7 Other proteins involved in the process of HIV-1 reverse transcription .................................................................................... 33 2.7.1 Viral proteins involved in reverse transcription ................... 33 2.7.2 Cellular proteins involved in HIV-1 reverse transcription ... 34 3. HIV-1 REVERSE TRANSCRIPTASE INHIBITORS .......................... 35 3.1 Nucleoside (nucleotide) reverse transcriptase inhibitors (NRTI) .. 35 3.1.1 General information ............................................................. 35 3.1.2 Life and fate of NRTI inside an infected cell ....................... 35 3.1.3 Resistance ............................................................................. 37 3.2 Non-nucleoside reverse transcriptase inhibitors (NNRTIs) .......... 38 5 3.2.1 General information ............................................................. 38 3.2.2 Chemical structure ................................................................ 39 3.2.3 The NNRTI binding pocket .................................................. 40 3.2.4 Mechanism of inhibition ...................................................... 41 3.2.5 Influence of NNRTI binding to particular steps of the reverse transcription reaction ............................................... 41 3.2.6 Inhibition of other stages of the viral life cycle by NNRTIs 42 3.2.7 Resistance ............................................................................. 42 STATE OF THE ART AND DEFINITION OF TASKS ............................ 44 AIMS OF THE CURRENT STUDY .......................................................... 46 RESULTS AND DISCUSSION ................................................................. 47 1. Development of a new screening system for testing the antiretroviral activities of compounds (unpublished) ........................ 47 2. Antiretroviral activities of novel acyclic thymine nucleoside analogues (article I) ........................................................................... 49 3. Antiretroviral activity of bimorpholines (article II and unpublished data) ................................................................................................... 51 4. Antiretroviral activity of saccharide hydrazones (article III) ........... 52 5. Non-nucleoside reverse transcriptase inhibitors and in silico screening (article iv) .......................................................................... 54 5.1 Cytotoxicity and antiretroviral activity of 16 compounds selected using in silico screening .............................................. 54 5.2 Chemical characteristics of compound 8. Antiretroviral activity of compound 8 (purified from the commercial sample) and its separated isomers ............................................. 55 5.3 Identification of the active component(s) from the initial sample of compound 8 .............................................................. 56 5.4 The analysis of the methods that were possibly used for the synthesis of compound 8 ........................................................... 57 5.5 The analysis of the x/y fraction using combined high-resolution MS and NMR approach ................................... 59 CONCLUSIONS ......................................................................................... 61 SUMMARY IN ESTONIAN ...................................................................... 63 REFERENCES ............................................................................................ 65 ACKNOWLEDGEMENTS ........................................................................ 84 PUBLICATIONS ........................................................................................ 85 CURRICULUM VITAE ............................................................................. 167 6 LIST OF ORIGINAL PUBLICATIONS This thesis is based on following publications, that are referred to by their Roman numerals in the text: I. Paju A, Päri M, Selyutina A, Zusinaite E, Merits A, Pehk T, Siirde K, Müüri- sepp AM, Kailas T, Lopp M. (2010). Synthesis of novel acyclic nucleoside analogues with anti-retroviral activity. Nucleosides Nucleotides Nucleic Acids, 29(9):707–720. II. Ausmees K*, Selyutina A*, Kütt K, Lippur K, Pehk T, Lopp M, Zusinaite E, Merits A, Kanger T. (2011). Synthesis and biological activity of bimorpholine and its carbanucleoside. Nucleosides Nucleotides Nucleic Acids, 30(11): 897–907. III. Ilisson M, Tomson K, Selyutina A, Türk S & Uno Mäeorg. (2015). Synthe- sis of Novel Saccharide Hydrazones. Synthetic Communications: An International Journal for Rapid Comunication of Synthetic Organic Chemistry, 45 (11): 1367–1373 IV. Viira B*, Selyutina A*, García-Sosa AT*, Karonen M, Sinkkonen J, Merits A, Maran U. Design, discovery, modeling, synthesis, and analysis of low tox- icity, novel s-triazine derivatives as HIV-1 non-nucleoside reverse transcriptase inhibitors. Under revision *These authors contributed equally to this work. This thesis also contains unpublished data Author’s contributions: I. I performed toxicity tests on HeLa cells, as well as tests for anti-HIV and anti- HCV activity. I analyzed the data and wrote the biological portions of the manuscript. II. I performed tests for toxicity, as well as anti-HIV and anti-HCV activity. I analyzed the data and wrote the biological portions of the manuscript. III. I designed and performed toxicity and antiviral activity tests. I analyzed the data and took part in writing the manuscript. IV. I designed and performed all of the biological experiments. I analyzed the data and wrote the biological portions of the manuscript. 7 LIST OF ABBREVIATIONS 3TC lamivudine aa amino acids (residues) ABC abacavir AIDS acquired immunodefficiency syndrome APOBEC apolipoprotein B mRNA-editing enzyme catalytic polypeptide ART antiretroviral therapy Att region attachment region AZT azidothymidine CA HIV-1 capsid protein CC50 50% cytotoxic concentration CCR5 C-C chemokine receptor type 5 CD cluster of differentiation CRF circulating recombinant form CXCR4 C-X-C chemokine receptor type 4 d4T stavudine ddC, ddCTP dideoxycytidine ddI didanosine DDX3 DEAD box helicase 3 DIS dimerization initiation signal DMSO dimethyl sulfoxide (c) (v) DNA (complementary) (viral) deoxyribonucleic acid DSIF DRB (5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity inducing factor EGFP enhanced green fluorescent protein Env envelope protein ESCRT endosomal sorting complex required for transport FDA Food and Drug Administration of the USA FSS frameshit stimulatory signal FTC emtricitabine Gag group-specific antigen gp glycoprotein HCV hepatitis C virus HIV-1 human immunodefficiency virus type-1 HLA human leukocyte antigen HPLC high-performance liquid chromatography Hsp70 heat shock protein 70 HSV-1 herpes simplex virus type-1 IC50 50% inhibitory concentration ICAM-1 intercellular adhesion molecule 1 IN HIV-1 integrase IRES internal ribosome entry site LTR long terminal repeat 8 MA HIV-1 matrix protein MRP multidrug resistance protein MS mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NC HIV-1 nucleocapsid protein Nef negative factor NELF negative elongation factor NMR nuclear magnetic resonance NNRTI non-nucleoside reverse transcriptase inhibitor NPC nuclear pore complex N site nucleotide binding site nt nucleotide (residue) N(t)RTI nucleoside (nucleotide) reverse transcriptase inhibitor p6 HIV-1 peptide 6 p51, p66 HIV-1 protein 51, 66 (reverse transcriptase subunits) PDB ID protein data bank identifier PBS primer-binding site PIC pre-integration complex PM plasma membrane PMA 12-myristate 13-acetate Pol HIV-1 polymerase (reverese transcriptase) Poly(A) polyadenilation signal PPi pyrophosphate (c, 3’) PPT (central, 3’) polypurine tract PR HIV-1 protease P site priming site P-TEFb positive transcription elongation factor b R region redundant region Rev regulator of expression of virion proteins RHA RNA helicase A RNase H ribonuclease H RNP ribonucleoprotein RT reverse transcriptase, reverse transcription RTC reverse transcription complex RRE Rev-responsive element SI selectivity index SIV Simian immunodeficiency virus SU HIV1-antireceptor (surface unit) TAR trans-acting responsive element Tat trans-activator of transcription TDF tenofovir disoproxil fumarate TFV tenofovir TK1 thymidine kinase 1 TM HIV-1 fusion protein (transmembrane protein) 9 TP triphosphates U5 (U3) region unique region for 5’-end (3’-end) UNG uracil DNA glycosylase UTR untranslated region Vif virion infectivity factor VLP virus-like particle Vpr viral protein R Vpu viral protein U WHO World Health Organization 10